Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activity

The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo‐distal axis of the branchial arches in mice. This pattern is known as the “Dlx code” and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis‐ regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates.     

Spatio‐temporal dynamics of gene expression of the Edn1‐Dlx5/6 pathway during development of the lower jaw

The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin‐1 (Edn1)‐dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo‐mandibular identity. Here, to better analyze the spatio‐temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1‐dependent and ‐independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1→Dlx5/6→Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events. genesis 48:362–373, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates

In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes.     

Expression and function of Dlx genes in the osteoblast lineage

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5^−/−/Dlx6^−/− mice have more severe craniofacial and limb defects than Dlx5^−/−, some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5^−/− mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.     

Functional conservation of a forebrain enhancer from the elephant shark (<Emphasis Type="Italic">Callorhinchus milii</Emphasis>) in zebrafish and mice

Background  

The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse.     

Dlx homeobox gene family expression in osteoclasts

Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time‐ and site‐specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts. J. Cell. Physiol. 223:779–787, 2010. © 2010 Wiley‐Liss, Inc.     

Msx1 and Dlx5 function synergistically to regulate frontal bone development

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1^?/?;Dlx5^?/? mice, the frontal bones defect was more severe than that of either Msx1^?/? or Dlx5^?/? mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development. genesis 48:645–655, 2010. © 2010 Wiley‐Liss, Inc.     

Retinoic acid affects craniofacial patterning by changing Fgf8 expression in the pharyngeal ectoderm

Retinoic acid signaling plays important roles in establishing normal patterning and cellular differentiation during embryonic development. In this study, we show that single administration of retinoic acid at embryonic day 8.5 causes homeotic transformation of the lower jaw into upper jaw-like structures. This homeosis was preceded by downregulation of Fgf8 and Sprouty expression in the proximal domain of the first pharyngeal arch. Downregulation of mesenchymal genes such as Dlx5, Hand2, Tbx1 and Pitx2 was also observed. The oropharynx in retinoic acid-treated embryos was severely constricted. Consistent with this observation, Patched expression in the arch endoderm and mesenchyme was downregulated. Thus, retinoic acid affects the expression of subsets of epithelial and mesenchymal genes, possibly disrupting the regional identity of the pharyngeal arch.     

Genomic Analysis of a New Mammalian Distal-less Gene: Dlx7

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.     

The Dlx genes as clues to vertebrate genomics and craniofacial evolution

The group of Dlx genes belongs to the homeobox-containing superfamily, and its members are involved in various morphogenetic processes. In vertebrate genomes, Dlx genes exist as multiple paralogues generated by tandem duplication followed by whole genome duplications. In this review, we provide an overview of the Dlx gene phylogeny with an emphasis on the chordate lineage. Referring to the Dlx gene repertoire, we discuss the establishment and conservation of the nested expression patterns of the Dlx genes in craniofacial development. Despite the accumulating genomic sequence resources in diverse vertebrates, embryological analyses of Dlx gene expression and function remain limited in terms of species diversity. By supplementing our original analysis of shark embryos with previous data from other osteichthyans, such as mice and zebrafish, we support the previous speculation that the nested Dlx expression in the pharyngeal arch is likely a shared feature among all the extant jawed vertebrates. Here, we highlight several hitherto unaddressed issues regarding the evolution and function of Dlx genes, with special reference to the craniofacial development of vertebrates.     

Expression of the Distalless-B gene in Ciona is regulated by a pan-ectodermal enhancer module

The Ci-Dll-B gene is an early regulator of ectodermal development in the ascidian Ciona intestinalis (Imai et al., 2006). Ci-Dll-B is located in a convergently transcribed bigene cluster with a tandem duplicate, Ci-Dll-A. This clustered genomic arrangement is the same as those of the homologous vertebrate Dlx genes, which are also arranged in convergently transcribed bigene clusters. Sequence analysis of the C. intestinalis Dll-A-B cluster reveals a 378 bp region upstream of Ci-Dll-B, termed B1, which is highly conserved with the corresponding region from the congener Ciona savignyi. The B1 element is necessary and sufficient to drive expression of a lacZ reporter gene in a pattern mimicking the endogenous expression of Ci-Dll-B at gastrula stages. This expression pattern which is specific to the entire animal hemisphere is activated preferentially in posterior, or b-lineage, cells by a central portion of B1. Expression in anterior, or a-lineage cells, can be activated by this central portion in combination with the distal part of B1. Anterior expression can also be activated by the central part of B1 plus both the proximal part of B1 and non-conserved sequence upstream of B1. Thus, cis-regulation of early Ci-Dll-B expression is activated by a required submodule in the center of B1, driving posterior expression, which works in combination with redundant submodules that respond to differentially localized anterior factors to produce the total animal hemisphere expression pattern. Interestingly, the intergenic region of the cluster, which is important for expression of the Dlx genes in vertebrates, does not have a specific activating function in the reporter genes tested, but acts as an attenuator in combination with upstream sequences.     

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.     

Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling

Background: Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial–mesenchymal interactions. METHODS: This study investigates the role of Dlx5 in the epithelial–mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high‐density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial–mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium. RESULTS: Dlx5 deletion alters expression of TGFβ₁, important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA‐treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects. CONCLUSIONS : Disruption in an Fgf3, ‐10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA. Birth Defects Res (Part B) 2008. ©2008 Wiley‐Liss, Inc.