DLX Genes in the Development and Maintenance of the Vertebrate Skeleton: Implications for Human Pathologies.

Skeletal shape and mechanical properties define, to a large extent, vertebrate morphology and physical capacities. During development, skeletal morphogenesis results from dynamic communications between chondrocytes, osteoblasts, osteoclasts, and other cellular components of the skeleton. Later in life, skeletal integrity depends on the regulatory cascades that assure the equilibrium between bone formation and resorption. Finally, during aging, skeletal catabolism prevails over anabolism resulting in progressive skeletal degradation. These cellular processes depend on the transcriptional cascades that control cell division and differentiation in each cell type. Most Distal-less (Dlx) homeobox transcription factors are directly involved in determining the proliferation and differentiation of chondrocytes and osteoblasts and, indirectly, of osteoclasts. While the involvement of Dlx genes in the regulation of skeletal formation has been well-analyzed thanks to several mutant mouse models, the role of these genes in the maintenance of bone integrity has been only partially studied. The importance of Dlx genes for adult bone tissues is evidenced by their central role in the regulatory pathways involving Osx/Sp7 and Runx2, the two major master genes of osteogenesis. Dlx genes appear to be involved in several bone pathologies including, for example, osteoporosis. Indeed, at least five large-scale GWAS studies which aimed to detect loci associated with human bone mineral density (BMD) have identified a known DLX5/6 regulatory region within chromosome 7q21.3 in proximity of SEM1/FLJ42280/DSS1 coding sequences, suggesting that DLX5/6 expression is critical in determining healthy BMD. This review aims to summarize the major findings concerning the involvement of Dlx genes in skeletal development and homeostasis and their involvement in skeletal aging and pathology.

Distal-less homeobox genes Dlx5/6 regulate Müllerian duct regression.

Dlx5 and Dlx6 encode distal-less homeodomain transcription factors that are present in the genome as a linked pair at a single locus. Dlx5 and Dlx6 have redundant roles in craniofacial, skeletal, and uterine development. Previously, we performed a transcriptome comparison for anti-Müllerian hormone (AMH)-induced genes expressed in the Müllerian duct mesenchyme of male and female mouse embryos. In that study, we found that Dlx5 transcripts were nearly seven-fold higher in males compared to females and Dlx6 transcripts were found only in males, suggesting they may be AMH-induced genes. Therefore, we investigated the role of Dlx5 and Dlx6 during AMH-induced Müllerian duct regression. We found that Dlx5 was detected in the male Müllerian duct mesenchyme from E14.5 to E16.5. In contrast, in female embryos Dlx5 was detected in the Müllerian duct epithelium. Dlx6 expression in Müllerian duct mesenchyme was restricted to males. Dlx6 expression was not detected in female Müllerian duct mesenchyme or epithelium. Genetic experiments showed that AMH signaling is necessary for Dlx5 and Dlx6 expression. Müllerian duct regression was variable in Dlx5 homozygous mutant males at E16.5, ranging from regression like controls to a block in Müllerian duct regression. In E16.5 Dlx6 homozygous mutants, Müllerian duct tissue persisted primarily in the region adjacent to the testes. In Dlx5-6 double homozygous mutant males Müllerian duct regression was also found to be incomplete but more severe than either single mutant. These studies suggest that Dlx5 and Dlx6 act redundantly to mediate AMH-induced Müllerian duct regression during male differentiation.

Dlx5/6 Expression Levels in Mouse GABAergic Neurons Regulate Adult Parvalbumin Neuronal Density and Anxiety/Compulsive Behaviours.

Neuronal circuits integrating Parvalbumin-positive GABAergic inhibitory interneurons (PV) are essential for normal brain function and are often altered in psychiatric conditions. During development, Dlx5 and Dlx6 (Dlx5/6) genes are involved in the differentiation of PV-interneurons. In the adult, Dlx5/6 continue to be expressed at low levels in most telencephalic GABAergic neurons, but their importance in determining the number and distribution of adult PV-interneurons is unknown. Previously, we have shown that targeted deletion of Dlx5/6 in mouse GABAergic neurons (Dlx5/6VgatCre mice) results in altered behavioural and metabolic profiles. Here we evaluate the consequences of targeted Dlx5/6 gene dosage alterations in adult GABAergic neurons. We compare the effects on normal brain of homozygous and heterozygous (Dlx5/6VgatCre and Dlx5/6VgatCre/+ mice) Dlx5/6 deletions to those of Dlx5 targeted overexpression (GABAergicDlx5/+ mice). We find a linear correlation between Dlx5/6 allelic dosage and the density of PV-positive neurons in the adult prelimbic cortex and in the hippocampus. In parallel, we observe that Dlx5/6 expression levels in GABAergic neurons are also linearly associated with the intensity of anxiety and compulsivity-like behaviours. Our findings reinforce the notion that regulation of Dlx5/6 expression is involved in individual cognitive variability and, possibly, in the genesis of certain neuropsychiatric conditions.

DLX5/6 GABAergic Expression Affects Social Vocalization: Implications for Human Evolution.

DLX5 and DLX6 are two closely related transcription factors involved in brain development and in GABAergic differentiation. The DLX5/6 locus is regulated by FoxP2, a gene involved in language evolution and has been associated with neurodevelopmental disorders and mental retardation. Targeted inactivation of Dlx5/6 in mouse GABAergic neurons (Dlx5/6VgatCre mice) results in behavioral and metabolic phenotypes notably increasing lifespan by 33%. Here, we show that Dlx5/6VgatCre mice present a hyper-vocalization and hyper-socialization phenotype. While only 7% of control mice emitted more than 700 vocalizations/10 min, 30% and 56% of heterozygous or homozygous Dlx5/6VgatCre mice emitted more than 700 and up to 1,400 calls/10 min with a higher proportion of complex and modulated calls. Hyper-vocalizing animals were more sociable: the time spent in dynamic interactions with an unknown visitor was more than doubled compared to low-vocalizing individuals. The characters affected by Dlx5/6 in the mouse (sociability, vocalization, skull, and brain shape…) overlap those affected in the "domestication syndrome". We therefore explored the possibility that DLX5/6 played a role in human evolution and "self-domestication" comparing DLX5/6 genomic regions from Neanderthal and modern humans. We identified an introgressed Neanderthal haplotype (DLX5/6-N-Haplotype) present in 12.6% of European individuals that covers DLX5/6 coding and regulatory sequences. The DLX5/6-N-Haplotype includes the binding site for GTF2I, a gene associated with Williams-Beuren syndrome, a hyper-sociability and hyper-vocalization neurodevelopmental disorder. The DLX5/6-N-Haplotype is significantly underrepresented in semi-supercentenarians (>105 years of age), a well-established human model of healthy aging and longevity, suggesting their involvement in the coevolution of longevity, sociability, and speech.

Dlx5 and Dlx6 expression in GABAergic neurons controls behavior, metabolism, healthy aging and lifespan.

Dlx5 and Dlx6 encode two homeobox transcription factors expressed by developing and mature GABAergic interneurons. During development, Dlx5/6 play a role in the differentiation of certain GABAergic subclasses. Here we address the question of the functional role of Dlx5/6 in the mature central nervous system. First, we demonstrate that Dlx5 and Dlx6 are expressed by all subclasses of adult cortical GABAergic neurons. Then we analyze VgatΔDlx5-6 mice in which Dlx5 and Dlx6 are simultaneously inactivated in all GABAergic interneurons. VgatΔDlx5-6 mice present a behavioral pattern suggesting reduction of anxiety-like behavior and obsessive-compulsive activities, and a lower interest in nest building. Twenty-month-old VgatΔDlx5-6 animals have the same size as their normal littermates, but present a 25% body weight reduction associated with a marked decline in white and brown adipose tissue. Remarkably, both VgatΔDlx5-6/+ and VgatΔDlx5-6 mice present a 33% longer median survival. Hallmarks of biological aging such as motility, adiposity and coat conditions are improved in mutant animals. Our data imply that GABAergic interneurons can regulate healthspan and lifespan through Dlx5/6-dependent mechanisms. Understanding these regulations can be an entry point to unravel the processes through which the brain affects body homeostasis and, ultimately, longevity and healthy aging.

Posterior axis formation requires Dlx5/Dlx6 expression at the neural plate border.

Neural tube defects (NTDs), one of the most common birth defects in human, present a multifactorial etiology with a poorly defined genetic component. The Dlx5 and Dlx6 bigenic cluster encodes two evolutionary conserved homeodomain transcription factors, which are necessary for proper vertebrate development. It has been shown that Dlx5/6 genes are essential for anterior neural tube closure, however their role in the formation of the posterior structures has never been described. Here, we show that Dlx5/6 expression is required during vertebrate posterior axis formation. Dlx5 presents a similar expression pattern in neural plate border cells during posterior neurulation of zebrafish and mouse. Dlx5/6-inactivation in the mouse results in a phenotype reminiscent of NTDs characterized by open thoracic and lumbar vertebral arches and failure of epaxial muscle formation at the dorsal midline. The dlx5a/6a zebrafish morphants present posterior NTDs associated with abnormal delamination of neural crest cells showing altered expression of cell adhesion molecules and defects of motoneuronal development. Our findings provide new molecular leads to decipher the mechanisms of vertebrate posterior neurulation and might help to gather a better understanding of human congenital NTDs etiology.

Probing the origin of matching functional jaws: roles of Dlx5/6 in cranial neural crest cells.

Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories.

Comparative analysis of molecular signatures suggests the use of gabapentin for the management of endometriosis-associated pain.

BACKGROUND: It has been repetitively shown that the transcription factors DLX5 and DLX6 are drastically downregulated in endometriotic lesions when compared with eutopic endometrium. These findings suggest that regulatory cascades involving DLX5/6 might be at the origin of endometriosis symptoms such as chronic pelvic pain (CPP). We have shown that inactivation of Dlx5 and Dlx5/6 in the mouse uterus results in an endometrial phenotype reminiscent of endometriosis. METHODS: We focused on genes that present a similar deregulation in endometriosis and in Dlx5/6-null mice in search of new endometriosis targets. RESULTS: We confirmed a strong reduction of DLX5 expression in endometriosis implants. We identified a signature of 30 genes similarly deregulated in human endometriosis implants and in Dlx5/6-null mouse uteri, reinforcing the notion that the downregulation of Dlx5/6 is an early event in the progress of endometriosis. CACNA2D3, a component of the α2δ family of voltage-dependent calcium channel complex, was strongly overexpressed both in mutant mouse uteri and in endometriosis implants, were also CACNA2D1 and CACNA2D2, other members of the α2δ family involved in nociception, are upregulated. CONCLUSION: Comparative analysis of gene expression signatures from endometriosis and mouse models showed that calcium channel subunits α2δ involved in nociception can be targets for the treatment of endometriosis-associated pain. CACNA2D3 has been associated with pain sensitization and heat nociception in animal models. In patients, CACNA2D3 variants were associated with reduced sensitivity to acute noxious stimuli. As α2δs were targets of gabapentinoid analgesics, the results suggested the use of these drugs for the treatment of endometriosis-associated pain. Indeed, recent small-scale clinical studies have shown that gabapentin could be effective in women with CPP. The findings of this study reinforce the need for a large definitive trial.

Disturbed Placental Imprinting in Preeclampsia Leads to Altered Expression of DLX5, a Human-Specific Early Trophoblast Marker.

BACKGROUND: Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. METHODS: We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. RESULTS: We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5, with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in trophoblasts in vitro. The transcriptional profile of such cells mimics the transcriptome of preeclamptic placentas. Pan-mammalian comparative analysis identified DLX5 as part of the human-specific regulatory network of trophoblast differentiation. CONCLUSIONS: Our analysis provides evidence of a true association among disturbed imprinting, gene expression, and preeclampsia. As a result of disturbed imprinting, the upregulated DLX5 affects trophoblast proliferation. Our in vitro model might fill a vital niche in preeclampsia research. Human-specific regulatory circuitry of DLX5 might help explain certain aspects of preeclampsia.

Dlx5 and Dlx6 control uterine adenogenesis during post-natal maturation: possible consequences for endometriosis.

Dlx5 and Dlx6 are two closely associated homeobox genes which code for transcription factors involved in the control of steroidogenesis and reproduction. Inactivation of Dlx5/6 in the mouse results in a Leydig cell defect in the male and in ovarian insufficiency in the female. DLX5/6 are also strongly expressed by the human endometrium but their function in the uterus is unknown. The involvement of DLX5/6 in human uterine pathology is suggested by their strong downregulation in endometriotic lesions and upregulation in endometrioïd adenocarcinomas. We first show that Dlx5/6 expression begins in Müllerian ducts epithelia and persists then in the uterine luminal and glandular epithelia throughout post-natal maturation and in the adult. We then use a new mouse model in which Dlx5 and Dlx6 can be simultaneously inactivated in the endometrium using a Pgr(cre/+) allele. Post-natal inactivation of Dlx5/6 in the uterus results in sterility without any obvious ovarian involvement. The uteri of Pgr(cre/+); Dlx5/6(flox/flox) mice present very few uterine glands and numerous abnormally large and branched invaginations of the uterine lumen. In Dlx5/6 mutant uteri, the expression of genes involved in gland formation (Foxa2) and in epithelial remodelling during implantation (Msx1) is significantly reduced. Furthermore, we show that DLX5 is highly expressed in human endometrial glandular epithelium and that its expression is affected in endometriosis. We conclude that Dlx5 and Dlx6 expression determines uterine architecture and adenogenesis and is needed for implantation. Given their importance for female reproduction, DLX5 and DLX6 must be regarded as interesting targets for future clinical research.

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids.

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.

Distinct effects of Hoxa2 overexpression in cranial neural crest populations reveal that the mammalian hyomandibular-ceratohyal boundary maps within the styloid process.

Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures.

Role of Foxl2 in uterine maturation and function.

Foxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women.

Etiology of craniofacial malformations in mouse models of blepharophimosis, ptosis and epicanthus inversus syndrome.

Blepharophimosis, ptosis, epicanthus-inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by narrow palpebral fissures and eyelid levator muscle defects. BPES is often associated to premature ovarian insufficiency (BPES type I). FOXL2, a member of the forkhead transcription factor family, is the only gene known to be mutated in BPES. Foxl2 is essential for maintenance of ovarian identity, but the developmental origin of the facial malformations of BPES remains, so far, unexplained. In this study, we provide the first detailed account of the developmental processes leading to the craniofacial malformations associated to Foxl2. We show that, during development, Foxl2 is expressed both by Cranial Neural Crest Cells (CNCCs) and by Cranial Mesodermal Cells (CMCs), which give rise to skeletal (CNCCs and CMCs) and muscular (CMCs) components of the head. Using mice in which Foxl2 is selectively inactivated in either CNCCs or CMCs, we reveal that expression of Foxl2 in CNCCs is essential for the development of extraocular muscles. Indeed, inactivation of Foxl2 in CMCs has only minor effects on muscle development, whereas its inactivation in CNCCs provokes a severe hypoplasia of the levator palpabrae superioris and of the superior and inferior oblique muscles. We further show that Foxl2 deletion in either CNCCs or CMCs prevents eyelid closure and induces subtle skeletal developmental defects. Our results provide new insights in the complex developmental origin of human BPES and could help to understand the origin of other ocular anomalies associated to this syndrome.

Facial dysplasia in wild chimpanzees.

At least 10% of the Sebitoli chimpanzee community of the Kibale National Park (Uganda) present a characteristic facial phenotype with flattened nose, reduced nostrils, and concave mid-face. Affected individuals do not present skin lesions, and also young infants are affected. We suggest, therefore, a congenital origin of this defect.

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.

BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Transitory expression of Dlx5 and Dlx6 in maxillary arch epithelial precursors is essential for upper jaw morphogenesis.

Asymmetric, articulated jaws support active predation in vertebrates; they derive from the first pharyngeal arch (PA1) which generates both maxillary and mandibular components. PA1 is colonized by cranial neural crest cells (CNCCs) which give rise to most bones and tendons of the jaws. The elements formed by different CNCCs contingents are specified by the combinatorial expression of Dlx genes. Dlx5 and Dlx6 are predominantly expressed by mandibular CNCCs. Analysis of the phenotype of Dlx5 and Dlx6 double mutant mice has suggested that they are necessary and sufficient to specify mandibular identity. Here, using 3D reconstruction, we show that inactivation of Dlx5 and Dlx6 does not only affect the mandibular arch, but results in the simultaneous transformation of mandibular and maxillary skeletal elements which assume a similar morphology with gain of symmetry. As Dlx5- and Dlx6-expressing cells are not found in the maxillary bud, we have examined the lineage of Dlx5-expressing progenitors using an in vivo genetic approach. We find that a contingent of cells deriving from precursors transiently expressing Dlx5 participate in the formation of the maxillary arch. These cells are mostly located in the distal part of the maxillary arch and might derive from its lambdoidal junction with the olfactory pit. Our findings extend current models of jaw morphogenesis and provide an explanation for the maxillary defects of Dlx5 and Dlx6 mutants. Our results imply that Dlx5 and Dlx6 model the upper and the lower PA1 components through different morphogenetic mechanisms which are, however, coordinated as they give rise to functional, articulated jaws.

Genome-wide linkage in a highly consanguineous pedigree reveals two novel loci on chromosome 7 for non-syndromic familial Premature Ovarian Failure.

BACKGROUND: The human condition known as Premature Ovarian Failure (POF) is characterized by loss of ovarian function before the age of 40. A majority of POF cases are sporadic, but 10-15% are familial, suggesting a genetic origin of the disease. Although several causal mutations have been identified, the etiology of POF is still unknown for about 90% of the patients. METHODOLOGY/PRINCIPAL FINDINGS: We report a genome-wide linkage and homozygosity analysis in one large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance. We identified two regions with a LOD(max) of 3.26 on chromosome 7p21.1-15.3 and 7q21.3-22.2, which are supported as candidate regions by homozygosity mapping. Sequencing of the coding exons and known regulatory sequences of three candidate genes (DLX5, DLX6 and DSS1) included within the largest region did not reveal any causal mutations. CONCLUSIONS/SIGNIFICANCE: We detect two novel POF-associated loci on human chromosome 7, opening the way to the identification of new genes involved in the control of ovarian development and function.

Masticatory muscle defects in hemifacial microsomia: a new embryological concept.

First arch syndromes correspond to a wide spectrum of human latero-facial congenital anomalies affecting cranial neural crest cells (CNCCs) derivatives of the first pharyngeal arch (PA1). The abnormal traits display variable quantitative expression and are often unilateral. Mandibular skeletal defects are invariably accompanied by hypoplasia or agenesis of masticatory muscles, but no explanation has been proposed for this association. Indeed, during embryonic development, CNCCs give only rise to skeletal components of the head while muscles derive from cephalic myogenic mesodermal cells (CMMCs). Recent studies on animal models have shown that communication between CNCCs and CMMCs is essential for the development of masticatory muscles: genetic lesions affecting only CNCCs can prevent muscularization of the jaws. To evaluate the involvement of CNCC/CMMC interactions in human craniofacial development, we performed a quantitative analysis of masticatory muscle and mandibular bone volumes on craniofacial CT-scans from 8 children, ages 3 months to 16 years, affected by hemifacial microsomia. We found that: (1) in seven patients the masseter muscle is absent in the affected side; (2) the absence of masseter is correlated neither with the age of the patients nor with the volume and shape of the affected ramus; and (3) in all cases the pterygoid and the temporal muscles are either reduced or absent. Our findings suggest that an early developmental event is the origin of the muscular defects in these patients. We propose that the hypoplasia or agenesis of masticatory muscles derives from a defect in the CNCCs/CMMCs communication during early embryonic development.