[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(1):e51700. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The congenital malformation Split Hand-Foot Malformation (SHFM, or ectrodactyly) is characterized by a medial cleft of hands and feet, and missing central fingers. Five genetically distinct forms are known in humans; the most common (type-I) is linked to deletions of DSS1 and the distalless-related homeogenes DLX5 and DLX6. As Dlx5;Dlx6 double-knockout mice show a SHFM-like phenotype, the human orthologs are believed to be the disease genes. SHFM-IV and Ectrodactyly-Ectodermal dysplasia-Cleft lip (EEC) are caused by mutations in p63, an ectoderm-specific p53-related transcription factor. The similarity in the limb phenotype of different forms of SHFM may underlie the existence of a regulatory cascade involving the disease genes. Here, we show that p63 and Dlx proteins colocalize in the nuclei of the apical ectodermal ridge (AER). In homozygous p63- (null) and p63EEC (R279H) mutant limbs, the AER fails to stratify and the expression of four Dlx genes is strongly reduced; interestingly, the p63+/EEC and p63+/- hindlimbs, which develop normally and have a normally stratified AER, show reduced Dlx gene expression. The p63+/EEC mutation combined with an incomplete loss of Dlx5 and Dlx6 alleles leads to severe limb phenotypes, which are not observed in mice with either mutation alone. In vitro, DeltaNp63alpha induces transcription from the Dlx5 and Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations, but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) mutations. ChIP analysis shows that p63 is directly associated with the Dlx5 and Dlx6 promoters. Thus, our data strongly implicate p63 and the Dlx5-Dlx6 locus in a pathway relevant in the aetio-pathogenesis of SHFM.
Development 05/2008; 135(7):1377-88. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Development of the olfactory pathway requires interaction between cells and signals of different origin. Olfactory receptor
neurons (ORN) in the olfactory placodes (OP) extend axons towards the forebrain (FB); with innervation taking place at a later
time following degradation of the basement membrane. Cells from the OP migrate along ORN axons and differentiate into various
elements, including ensheathing and Gonadotropin Releasing Hormone (GnRH)+ cells. The importance of the olfactory connection
and migration is highlighted by the severe endocrine phenotype in Kallmann’s patients who lack this migratory pathway. Little
is known about the genetic control of intrinsic ORN properties. Inactivation of the distalless-related Dlx5 prevents connections between ORNs and FB. Using a grafting approach we show that misguidance and lack of connectivity is
due to intrinsic defects in ORN neurites and migratory cells (MgC), and not to environmental factors. These data point to
a cell-autonomous function of Dlx5 in providing ORN axons with their connectivity properties. Dlx5 also marks a population of early MgC that partly overlaps with the GnRH+ population. In the absence of Dlx5 MgCs of the Dlx5+ lineage migrate, associated with PSA-NCAM+ axons, but fail to reach the FB as a consequence of the lack
of axonal connection and not an inability to migrate. These data suggests that Dlx5 is not required to initiate migration and differentiation of MgCs.
Journal of Molecular Histology 01/2008; 38(6):612-23. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: A variety of signals governing early extension, guidance, and connectivity of olfactory receptor neuron (ORN) axons has been identified; however, little is known about axon-mesoderm and forebrain (FB)-mesoderm signals. Using Wnt-beta catenin reporter mice, we identify a novel Wnt-responsive resident cell population, located in a Frizzled7 expression domain at the surface of the embryonic FB, along the trajectory of incoming ORN axons. Organotypic slice cultures that recapitulate olfactory-associated Wnt-beta catenin activation show that the beta catenin response depends on a placode-derived signal(s). Likewise, in Dlx5-/- embryos, in which the primary connections fail to form, Wnt-beta catenin response on the surface of the FB is strongly reduced. The olfactory placode expresses a number of beta catenin-activating Wnt genes, and the Frizzled7 receptor transduces the "canonical" Wnt signal; using Wnt expression plasmids we show that Wnt5a and Wnt7b are sufficient to rescue beta catenin activation in the absence of incoming axons. Finally, blocking the canonical Wnt pathway with the exogenous application of the antagonists Dikkopf-1 or secreted-Frizzled-receptor protein-2 prevents ORN axon contact to the FB. These data reveal a novel function for Wnt signaling in the establishment of periphery-CNS olfactory connections and highlight a complex interplay between cells of different embryonic origin for ORN axon connectivity.
Journal of Neuroscience 10/2007; 27(36):9757-68. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.
Mechanisms of Development 02/2006; 123(1):3-16. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: CTLA-4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA-4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA-4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast-like cultures). However, by reverse transcriptase-PCR, CTLA-4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA-4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA-4-expressing tumor lines with recombinant forms of the CTLA-4-ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase-8 and caspase-3. The level of apoptosis was reduced by soluble CTLA-4 and by anti-CTLA-4 scFvs antibodies. The novel finding that CTLA-4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.
International Journal of Cancer 12/2005; 117(4):538-50. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Charcot-Marie-Tooth type 1A (CMT1A) is a hereditary demyelinating neuropathy due to an increased genetic dosage of the peripheral myelin protein 22 (PMP22). The mechanisms leading from PMP22 overexpression to impairment of myelination are still unclear. We evaluated expression and processing of PMP22, viability, proliferation, migration, motility and shaping properties, and ability of forming myelin of PMP22 transgenic (PMP22(tg)) Schwann cells in culture. In basal conditions, PMP22(tg) Schwann cells, although expressing higher PMP22 levels than control ones, show normal motility, migration and shaping properties. Addition of forskolin to the media induces an additional stimulation of PMP22 expression and results in an impairment of cells migration and motility, and a reduction of cell area and perimeter. Similarly, co-culturing transgenic Schwann cells with neurons causes an altered cells differentiation and an impairment of myelin formation. In conclusion, exposure of PMP22(tg) Schwann to the axon or to axonal-mimicking stimuli significantly affects the transition of transgenic Schwann cells to the myelinating phenotype.
Neurobiology of Disease 07/2004; 16(1):263-73. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: The distalless-related homeogene Dlx5 is expressed in the olfactory placodes and derived tissues and in the anterior-basal forebrain. We investigated the role of Dlx5 in olfactory development. In Dlx5(-/-) mice, the olfactory bulbs (OBs) lack glomeruli, exhibit disorganized cellular layers, and show reduced numbers of TH- and GAD67-positive neurons. The olfactory epithelium in Dlx5(-/-) mice is composed of olfactory receptor neurons (ORNs) that appear identical to wild-type ORNs, but their axons fail to contact the OBs. We transplanted Dlx5(-/-) OBs into a wild-type newborn mouse; wild-type ORN axons enter the mutant OB and form glomeruli, but cannot rescue the lamination defect or the expression of TH and GAD67. Thus, the absence of Dlx5 in the OB does not per se prevent ORN axon ingrowth. In conclusion, Dlx5 plays major roles in the connectivity of ORN axons and in the differentiation of OB interneurons.
Molecular and Cellular Neuroscience 05/2003; 22(4):530-43. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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) was drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. We propose 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 BPM4 expression and the regional organization of cell differentiation, proliferation, and apoptosis.
[show abstract][hide abstract] ABSTRACT: Split hand/foot malformation type I (SHFM1) disease locus maps to chromosome 7q21.3-q22, a region that includes the distal-less-related (dll) genes DLX5 and DLX6. However, incomplete penetrance, variable expressivity, segregation distortion, and syndromic association with other anomalies have so far prevented the identification of the SHFM1 gene(s) in man. Here we show that the targeted double inactivation of Dlx5 and Dlx6 in the mouse causes in homozygous mutant animals bilateral ectrodactyly with a severe defect of the central ray of the hindlimbs, a malformation typical of SHFM1. This is the first evidence that the role of dll/Dlx genes in appendage development is conserved from insects to mammals and proves their involvement in SHFM1.
[show abstract][hide abstract] ABSTRACT: The subventricular zone of the adult mammalian brain harbors the neural stem cell population with potential neural regeneration and repair capacity. We describe a nonviral technique to preferentially transfect in vivo the adult neural stem cell population and its immediate progeny based on intraventricular injection of PEI/DNA complexes. The transfected population was identified by cellular and ultra-structural evidence showing their proliferating status and expression of the specific markers GFAP and nestin. Stable activation of the lacZ reporter by cre-recombinase transfection in R26R mice demonstrated survival and migration of stem cell derivatives three months after injection. Apoptosis is thought to be the most common fate of the stem cell progeny. Overexpression of Bcl-X(L) increased number and survival time of transduced progenitors and decreased the frequency of cells immunopositive for activated Caspase-3. This method thus provides selective targeting of the stem cell population and should allow an in-depth understanding of their biology.
Molecular and Cellular Neuroscience 03/2002; 19(2):165-74. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fgf4, a member of the fibroblast growth factor family, is frequently amplified in a variety of human cancers, however, its expression in neoplastic tissues is rarely detectable. This makes uncertain its involvement in tumour aetiology, although several in-vitro studies link Fgf4 overexpression to malignant transformation and metastatization of culture cells. We generated a transgenic mouse model in which the whey acidic protein (WAP) promoter directs expression of human Fgf4 to mammary tissues during late pregnancy and throughout lactation, with the purpose of studying the involvement of this growth factor in mammary tumorigenesis. Expression of the transgene was specifically detected in lobular-alveolar cells of lactating mammary glands that, by histological analysis, displayed hyperplastic areas and a disorganized structure. This was accompanied by an increased number of red blood cells and expression, in alveolar epithelial cells, of the vascular endothelial growth factor, which is absent in wild type controls. The most striking effect caused by FGF4 overexpression was on the remodelling of mammary tissue at the end of lactation. Indeed, transgenic animals showed a delayed involution of the gland due to a dramatic reduction in the overall number of apoptotic cells, which are normally present in the organ after weaning. Nevertheless, none of the animals examined developed neoplastic lesions of the mammary gland even after several pregnancies and at old age. Our work represents the first in-vivo demonstration of the anti-apoptotic and angiogenic properties of FGF4.
[show abstract][hide abstract] ABSTRACT: Intravenous administration could become a delivery route of choice for prophylactic and curative gene therapies on condition that genes cross the capillary barrier and reach target tissues without being degraded. We investigated the kinetics and process of transgene delivery through mouse lung capillaries following DNA complexation with linear polyethylenimine (L-PEI) and intravenous injection. Using digoxin-labeled DNA we followed the cellular localization of DNA at different times after injection and correlated these findings with cell markers and transgene expression. At 2 h after injection some DNA was still localized on the interior of the capillary lumen, but other complexes had already crossed the barrier and resulted in gene expression. At 24 h after injection most labeled DNA was localised in pulmonary cells, as was transgene expression. Only rarely was transgene expression found in endothelial cells, suggesting that the complexes cross the capillary barrier rapidly. Levels of caspase-1-like activity did not increase following transfection implying that L-PEI/DNA complexes are transported across cellular barriers by a non-damaging, physiological process, without causing inflammation. The high levels of expression of different transgenes in pneumocytes indicates that transport of L-PEI/DNA complexes through the endothelial barrier does not affect their transfection capacity. These findings open up new possibilities for gene delivery and its application to the lung.
[show abstract][hide abstract] ABSTRACT: Dlx genes comprise a highly conserved family of homeobox genes homologous to the distal-less (Dll) gene of Drosophila. They are thought to act as transcription factors. All Dlx genes are expressed in spatially and temporally restricted patterns in craniofacial primordia, basal telencephalon and diencephalon, and in distal regions of extending appendages, including the limb and the genital bud. Most of them are expressed during morphogenesis of sensory organs and during migration of neural crest cells and interneurons. In addition, Dlx5 and Dlx6 are expressed in differentiating osteoblasts. Gene targeting of Dlx1, Dlx2, Dlx3 and Dlx5 in the mouse germ-line has revealed functions in craniofacial patterning, sensory organ morphogenesis, osteogenesis and placental formation. However, no effect on limb development has yet been revealed from gene inactivation studies. A role for these genes in limb development is however suggested by the linkage of the Split Foot/Hand Malformation human syndrome to a region containing DLX5 and DLX6. As for most transcription factors, these genes seem to have multiple functions at different stages of development or in different tissues and cell types.
The International Journal of Developmental Biology 02/2000; 44(6):619-26. · 2.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Development 10/1999; 126(17):3795-809. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Generally, cationic vector-based intravenous delivery of DNA is hindered by interactions of positively charged complexes with serum proteins. However, if optimally formulated, cationic vectors can provide reasonable levels of transfection in the lung either by intravenous or intrapulmonary routes. We investigated the in vivo transfection capacity of a cationic polymer: linear, 22 kDa polyethylenimine. PEI/DNA complexes were formulated in 5% glucose and delivered into adult mice through the tail vein. Two marker genes were used, beta-galactosidase and luciferase. High levels of luciferase expression (10(7) RLU/mg protein) were found in the lung when DNA was complexed with PEI at a ratio of 4 nitrogen equivalents per DNA phosphate. Lower levels of transfection were found in the heart, spleen, liver and kidney. Expression was dose- and time-dependent in all tissues examined. In the lung, beta-galactosidase staining showed transgene expression in clusters of 10 or more pulmonary cells including the alveolar endothelium, squamous and great alveolar epithelial cells (type I and II pneumocytes) and septal cells. These findings indicate that the complexes pass the capillary barrier in the lung. Although the delivery mechanism requires elucidation, linear PEI has promise as a vector for intravenous transfer of therapeutic genes.