Distal-less Research Articles

The effects of lead on GABAergic interneurons in rodents.

Lead is a heavy metal that affects various systems and organs in the body, especially the nervous system. In this study, the in vivo and in vitro effects of lead on neurons were analyzed. We divided mouse pups into three groups based on the concentration of lead exposure: the control group, the low-dose group, and the high-dose group. Changes in behavior (measured by an open-field test and a tail suspension test), blood lead levels (measured by atomic absorption spectrophotometry), the number of GABAergic interneurons (measured by immunohistochemistry), gene expression (measured by qRT-PCR), and DNA methylation (measured by pyrosequencing) were determined in the three groups. The lead-exposed pups showed significantly higher blood lead levels ( p < 0.001). Lead exposure caused hyperactivity and reduced the body weight of the exposed mice compared with that of the controls. The lead-exposed groups showed significantly lower numbers of parvalbumin and neuropeptide Y interneurons and lower expression levels of distal-less homeobox ( Dlx) 1, 2, 5, and 6 genes in the cerebral cortex. To further clarify the mechanism of Dlx gene downregulation, we selected the GE6 cell line, which can differentiate into various subtypes of GABAergic interneurons, for in vitro experiments. We found that high levels of lead also inhibited the expression of Dlx 1/ 2/ 5/ 6 in vitro, but DNA methylation levels were not changed in the GE6 cell line. Furthermore, lead exposure significantly decreased the expression of Olig1 and Ki67 and increased that of Tubb3 in vitro. The present study revealed that lead exposure can alter behaviors, reduce the number of GABAergic interneurons, and change the expression of some important genes in neuronal cells.

MiR-539 mediates osteoblast mineralization by regulating Distal-less genes 2 in MC3T3-E1 cell line

AbstractmicroRNAs (miRNAs) play an important role in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still needs to be further cleared. Distal-less genes 2 (Dlx2) plays an important role in osteoblast differentiation. We have found that miR-539 was significantly downregulated and Dlx2 was found to be inversely correlated with miR-539 in MC3T3-E1 cell line during osteoblast mineralization. The overexpression of miR-539 significantly decreased the expression level of Dlx2 and suppressed the osteogenic marker gene expression level, alkaline phosphatase activity and matrix mineralization. Our study showed that miR-539 was a negative regulator in osteoblast mineralization and that the targeting of Dlx2 gene partly contributes to this inhibitory effect exerted by miR-539.

GABAergic Interneuron Differentiation in the Basal Forebrain Is Mediated through Direct Regulation of Glutamic Acid Decarboxylase Isoforms by Dlx Homeobox Transcription Factors.

GABA is the key inhibitory neurotransmitter in the cortex but regulation of its synthesis during forebrain development is poorly understood. In the telencephalon, members of the distal-less (Dlx) homeobox gene family are expressed in, and regulate the development of, the basal ganglia primodia from which many GABAergic neurons originate and migrate to other forebrain regions. The Dlx1/Dlx2 double knock-out mice die at birth with abnormal cortical development, including loss of tangential migration of GABAergic inhibitory interneurons to the neocortex (Anderson et al., 1997a). We have discovered that specific promoter regulatory elements of glutamic acid decarboxylase isoforms (Gad1 and Gad2), which regulate GABA synthesis from the excitatory neurotransmitter glutamate, are direct transcriptional targets of both DLX1 and DLX2 homeoproteins in vivo Further gain- and loss-of-function studies in vitro and in vivo demonstrated that both DLX1 and DLX2 are necessary and sufficient for Gad gene expression. DLX1 and/or DLX2 activated the transcription of both Gad genes, and defects in Dlx function disrupted the differentiation of GABAergic interneurons with global reduction in GABA levels in the forebrains of the Dlx1/Dlx2 double knock-out mouse in vivo Identification of Gad genes as direct Dlx transcriptional targets is significant; it extends our understanding of Dlx gene function in the developing forebrain beyond the regulation of tangential interneuron migration to the differentiation of GABAergic interneurons arising from the basal telencephalon, and may help to unravel the pathogenesis of several developmental brain disorders.SIGNIFICANCE STATEMENT GABA is the major inhibitory neurotransmitter in the brain. We show that Dlx1/Dlx2 homeobox genes regulate GABA synthesis during forebrain development through direct activation of glutamic acid decarboxylase enzyme isoforms that convert glutamate to GABA. This discovery helps explain how Dlx mutations result in abnormal forebrain development, due to defective differentiation, in addition to the loss of tangential migration of GABAergic inhibitory interneurons to the neocortex. Reduced numbers or function of cortical GABAergic neurons may lead to hyperactivity states such as seizures (Cobos et al., 2005) or contribute to the pathogenesis of some autism spectrum disorders. GABAergic dysfunction in the basal ganglia could disrupt the learning and development of complex motor and cognitive behaviors (Rubenstein and Merzenich, 2003).

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons.

Flightless-I governs cell fate by recruiting the SUMO isopeptidase SENP3 to distinct HOX genes

BackgroundDespite recent studies on the role of ubiquitin-related SUMO modifier in cell fate decisions, our understanding on precise molecular mechanisms of these processes is limited. Previously, we established that the SUMO isopeptidase SENP3 regulates chromatin assembly of the MLL1/2 histone methyltransferase complex at distinct HOX genes, including the osteogenic master regulator DLX3. A comprehensive mechanism that regulates SENP3 transcriptional function was not understood.ResultsHere, we identified flightless-I homolog (FLII), a member of the gelsolin family of actin-remodeling proteins, as a novel regulator of SENP3. We demonstrate that FLII is associated with SENP3 and the MLL1/2 complex. We further show that FLII determines SENP3 recruitment and MLL1/2 complex assembly on the DLX3 gene. Consequently, FLII is indispensible for H3K4 methylation and proper loading of active RNA polymerase II at this gene locus. Most importantly, FLII-mediated SENP3 regulation governs osteogenic differentiation of human mesenchymal stem cells.ConclusionAltogether, these data reveal a crucial functional interconnection of FLII with the sumoylation machinery that converges on epigenetic regulation and cell fate determination.

The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling.

Homeobox genes play a critical role in embryonic development, but they have also been implicated in cancer through mechanisms that are largely unknown. While not expressed during normal T-cell development, homeobox transcription factor genes can be reactivated via recurrent chromosomal rearrangements in human T-cell acute leukemia/lymphoma (T-ALL), a malignancy often associated with activated Notch and Akt signaling. To address how epigenetic reprogramming via an activated homeobox gene might contribute to T-lymphomagenesis, we investigated a transgenic mouse model with thymocyte-specific overexpression of the Dlx5 homeobox gene. We demonstrate for the first time that Dlx5 induces T-cell lymphomas with high penetrance. Integrated ChIP-seq and mRNA microarray analyses identified Notch1/3 and Irs2 as direct transcriptional targets of Dlx5, a gene signature unique to lymphomas from Lck-Dlx5 mice as compared to T-cell lymphomas from Lck-MyrAkt2 mice, which were previously reported by our group. Moreover, promoter/enhancer studies confirmed that Dlx5 directly transactivates Notch expression. Notch1/3 expression and Irs2-induced Akt signaling were upregulated throughout early stages of T-cell development, which promoted cell survival during β-selection of T lymphocytes. Dlx5 was required for tumor maintenance via its activation of Notch and Akt, as tumor cells were highly sensitive to Notch and Akt inhibitors. Together, these findings provide unbiased genetic and mechanistic evidence that Dlx5 acts as an oncogene when aberrantly expressed in T cells, and that it is a novel discovery that Notch is a direct target of Dlx5. These experimental findings provide mechanistic insights about how reactivation of the Dlx5 gene can drive T-ALL by aberrant epigenetic reprogramming of the T-cell genome.

Polymorphisms in genes involved in enamel development are associated with dental fluorosis

ObjectiveTo evaluate the association between polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 genes with dental fluorosis (DF) phenotype. DesignFour hundred and eighty one subjects (108 with DF and 373 DF free) from 6 to 18 years of age were recruited. This population lived in Rio de Janeiro, a city with fluoridation of public water supplies. DF was assessed using the Deańs index modified. Only erupted permanent teeth were assessed. Genetic polymorphisms in DLX1, DLX2, MMP13, TIMP1 and TIMP2 were analyzed by real-time PCR from genomic DNA. Association between DF, genotype, and allele distribution were evaluated using chi-square and logistic regression analyses with an alpha level of 5%. ResultsDF was more prevalent in Afro-descendants than in Caucasians (p=0.08; OR=1.83; CI 95%=1.18–2.82). Logistic regression analysis adjusted by the ethnicity demonstrated a statistical difference for TIMP1 genotype (p=0.033; OR=2.93, 95%CI, 1.09–7.90). When only the severer cases of DF were analyzed, polymorphisms in DLX1 and DLX2 were associated with DF (p<0.05). ConclusionOur results provided evidence that polymorphisms in TIMP1, DLX1 and DLX2 genes may be associated with DF phenotypes.

Regulation of Brn3b by DLX1 and DLX2 is required for retinal ganglion cell differentiation in the vertebrate retina.

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo. We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.

DLX1 Affects Cell Cycle and Proliferation of Myeloid Leukemia Cells In Vitro and In Vivo

The DLX homeodomain genes are part of the Drosophila distal-less family, originally identified in the forebrain of the developing mouse embryo. DLX1 gene is expressed also in the hematopoietic cells. Our previous data showed that patients with FLT3/ITD (internal tandem duplication) mutation representing about 35% of all acute myeloid leukemia (AML) cases have higher expression of DLX1 compared to non-FLT3/ITD AML patients. Further, FLT3 signaling was described to regulate DLX1 gene expression.In the current study we found an association between DLX1 expression level and overall survival using GEP data from TCGA database within the group of FLT3/ITD-positive patients. Worse overall survival was linked to lower expression of DLX1 (p=0.003, n=46) even though generally this group of patients is characterized by their higher DLX1 level. This association was also observed when all AML patients were analyzed (p=0.01, n=197). Since the role of DLX1 in the leukemogenic process is not yet described we aimed to characterize the phenotype of leukemic cells with different expression levels of DLX1 gene.We designed two functional shRNAs (pGhU6-sh1_DLX1 and pGhU6-sh2_DLX1, referred as sh1 and sh2) to downregulate DLX1 expression in MV4;11 (FLT3/ITD positive) leukemic cells, which present high endogenous level of DLX1, and a non-silenced control (pGhU6-NSC). In vitro studies showed that cells with silenced DLX1 were arrested in G0 phase of cell cycle (35%±4.8 (NSC) to 67.5%±2.2 (sh1; p <0.01) and 65.5%±2.5 (sh2; p <0.01); flow cytometry - Pyronin Y/ Hoechst 33342 staining) and had lower proliferative activity (trypan blue over the period of 10 passages). Moreover, cells with silenced DLX1 were less apoptotic (Annexin V/PI staining).Next, we studied the impact of DLX1 downregulation on leukemic cell infiltration in vivo using sub-lethally irradiated NSG (NOD SCID gamma) mice. We injected 1x106 silenced or non-silenced MV4;11 cells via tail vein (n=20, 6 mice/group, 2 control mice). After two weeks we measured the absolute number of MV4;11 cells by flow cytometry (CD33+/GFP+/DAPI-) in bone marrow (BM) and spleen (SP) of recipient mice. Absolute cell counts of leukemic cells with silenced DLX1 were 1.26x106 (sh1; p=0.015) and 2.89x106 (sh2; p<0.0001) vs NSC cells 0.52x106 in BM and 1.59x106 (p=0.036) and 14.2x106 (p=0.025) vs 0.34x106 in SP. To ensure that the increased cell numbers were not the result of enhanced homing of DLX1 silenced cells, we performed homing experiments. Control and DLX1 silenced cells were transplanted and the number of cells in BM and SP was determined 16 hours after transplantation. We observed no differences between the studied groups, indicating that DLX1 silencing does not affect the homing ability of MV4;11 cells. Moreover, we analyzed cell cycle in leukemic cells isolated from recipient mice three weeks after transplantation. In agreement with our in vitro results, leukemic cells with silenced DLX1 had higher percentage of cells arrested in G0 phase (48.6%±6.3 (sh1; p <0.001) and 80.9%±9.3 (sh2; p <0.001) vs 26.4%±4.8 (NSC) in SP). Overall, mice with silenced DLX1 presented worse fitness and bigger splenomegaly.Further, we investigated the signaling pathways which could lead to G0 arrest. Since DLX1 inhibits TGF-β pathway through direct interaction with SMAD4, a key downstream effector of TGF-β/BMP signaling, we studied the changes in target genes in our model cell lines. Expression of CUTL-1 (sh1 - 1.5-fold, sh2 - 2-fold change to NSC), PAI-1 (3.5-fold, 4.2-fold) and CDKN1C (1.3-fold, 2.3-fold) were significantly increased in DLX1 silenced cells. PAI-1 was shown to induce replicative senescence and CDKN1C is an inhibitor of cell cycle progression. While both targets disturb cell cycle and could be responsible for the phenotype we have observed, this hypothesis needs to be elucidated in future experiments.Altogether, our data demonstrate that dysregulation of DLX1 gene in leukemic cells changes the cell phenotype. Lower level of DLX1 gene leads to arrest in G0 phase which in vitro slows down the proliferation whereas in vivo it allows the cells to persist in spleen and BM. We hypothesize that the DLX1 silenced cells become more resistant to external effects which could then be reflected as a reduced survival observed in patients with low levels of DLX1.Supported by P304/12/2214. DisclosuresNo relevant conflicts of interest to declare.

Functional consequences of I56ii Dlx enhancer deletion in the developing mouse forebrain

Dlx homeobox genes encode a group of transcription factors that play an essential role during developmental processes including maintaining the differentiation, proliferation and migration of GABAergic interneurons. The Dlx1/2 and Dlx5/6 genes are expressed in the forebrain and are arranged in convergently transcribed bigene clusters, with I12a/I12b and I56i/I56ii cis-regulatory elements (CREs) located in the intergenic region of each cluster respectively. We have characterized the phenotypic consequences of deleting I56ii on forebrain development and spatial patterning of corridor cells that are involved in guiding thalamocortical projections. Here we report that deletion of I56ii impairs expression of Dlx genes and that of potential targets including Gad2 as well as striatal markers Islet1, Meis2, and Ebf1. In addition, I56ii deletion reduces both the binding of DLX2 in the Dlx5/Dlx6 intergenic region and the presence of H3K9Ac at the Dlx5/Dlx6 locus, consistent with the reduced expression of these genes. Deletion of I56ii reduces the expression of the ISLET1 and CTIP2 in the striatum and disrupts the number of parvalbumin and calretinin expressing cells in the adult somatosensory cortex of the ΔI56ii mice. These data suggest an important regulatory role for I56ii in the developing forebrain by means of a potential regulatory mechanism which may regulate the expression of Dlx genes, notably Dlx6 as well as the spatial patterning of the ventral telencephalon, including possibly corridor cells.

Hypoxia enhances osteogenic differentiation in retinoic acid-treated murine-induced pluripotent stem cells

Supplementary material is available for this article at 10.1007/s13770-016-9127-9 and is accessible for authorized users.

354 Elucidating the DLX3 interactome to identify the mechanisms through which it regulates skin differentiation

354 Elucidating the DLX3 interactome to identify the mechanisms through which it regulates skin differentiation

Unexpected identification of a recurrent mutation in the DLX3 gene causing amelogenesis imperfecta.

To identify the molecular genetic aetiology of a family with autosomal dominant amelogenesis imperfecta (AI). DNA samples were collected from a six-generation family, and the candidate gene approach was used to screen for the enamelin (ENAM) gene. Whole-exome sequencing and linkage analysis with SNP array data identified linked regions, and candidate gene screening was performed. Mutational analysis revealed a mutation (c.561_562delCT and p.Tyr188Glnfs*13) in the DLX3 gene. After finding a recurrent DLX3 mutation, the clinical phenotype of the family members was re-examined. The proband's mother had pulp elongation in the third molars. The proband had not hair phenotype, but her cousin had curly hair at birth. In this study, we identified a recurrent 2-bp deletional DLX3 mutation in a new family. The clinical phenotype was the mildest one associated with the DLX3 mutations. These results will advance the understanding of the functional role of DLX3 in developmental processes.

Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes.

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro-osteogenic and pro-inflammatory cues.

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200pos cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up‐regulated in CD200pos cells compared to CD200neg fraction. At the functional level, CD200pos cells were prone to mineralize the extra‐cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200pos cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down‐regulated. As dexamethasone has anti‐inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro‐inflammatory cytokines interleukin‐1β and tumour necrosis factor‐α increased CD200 membrane expression but down‐regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro‐inflammatory or pro‐osteogenic, CD200 expression was down‐regulated when nuclear‐factor (NF)‐κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro‐inflammatory cytokines through the same pathway: NF‐κB.

Role of Genes in Odontogenesis

With the discovery of the homeobox genes in craniofacial biology researchers across the globe have studied in depth the genetic patterning of the craniofacial region. With respect to craniofacial development ‐, Barx, Dlx, Gsc, Lim, Msx, Otx, Prx; part of the Hox cluster are important. Barx gene are strongly expressed only in the mesenchyme of the developing molars. Dlx gene expression is noted in the mandibular and maxillary arch ectomesenchyme. Msx genes are expressed in the area of epithelial mesenchymal interactions in the brachial arches in the area of future dentition and also expressed in the formation of skull, facial primordial and sense organs. Msx-1 is seen to be expressed in various stages of tooth formation i.e bud and cap stage of organogenesis. Lim genes which control morphogenesis of the first brachial arch, are expressed in the maxillo-mandibular ectomesenchyme. Prx gene expression is seen in the proximal portion of the mandibular arch. The role of hox genes in the morphogenesis of the jaws and the dentition is immense. Thus it has been proved beyond doubt that the genes have a major role in organogenesis than what human beings have ever envisaged. This review will give the scientific community an overview of all the genes affecting odontogenesis.

The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6-/- shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand.

The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5–DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered β-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology.

Lineage tracing of neuronal progenitor cells expressing dlx genes in the zebrafish brain

International Journal of Developmental NeuroscienceVolume 47, Issue Part_A p. 40-40 Article ISDN2014_0139: Lineage tracing of neuronal progenitor cells expressing dlx genes in the zebrafish brain Cynthia Solek, Corresponding Author Cynthia Solek n/a@.dne Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaE-mail address: mekker@uottawa.ca (C. Solek).Search for more papers by this authorShengrui Feng, Shengrui Feng Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this authorElyssa Mahoney, Elyssa Mahoney Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this authorMarc Ekker, Marc Ekker mekker@uottawa.ca Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this author Cynthia Solek, Corresponding Author Cynthia Solek n/a@.dne Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaE-mail address: mekker@uottawa.ca (C. Solek).Search for more papers by this authorShengrui Feng, Shengrui Feng Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this authorElyssa Mahoney, Elyssa Mahoney Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this authorMarc Ekker, Marc Ekker mekker@uottawa.ca Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5 CanadaSearch for more papers by this author First published: 05 November 2015 https://doi.org/10.1016/j.ijdevneu.2015.04.114Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume47, IssuePart_AISDN 2014 AbstractsDecember 2015Pages 40-40 RelatedInformation

Expression of the Drosophila homeobox gene, Distal-less, supports an ancestral role in neural development.

Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos, we show that Dll is expressed in the embryonic, larval and adult central nervous system and peripheral nervous system (PNS) in embryonic and larval neurons, brain and ventral nerve cord glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts.