Distal-less Research Articles

Evolution of the forebrain — revisiting the pallium

Evolution of the forebrain — revisiting the pallium

Heterogeneous Conservation of Dlx Paralog Co-Expression in Jawed Vertebrates

BackgroundThe Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model.ResultsThe genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family.Conclusion Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.

Analysis of Microsatellite Polymorphisms in South Indian Patients with Non Syndromic Cleft Lip and Palate

Non syndromic cleft lip and/or palate (NSCLP) is a complex congenital anomaly with varying incidence among patients of different geographical origins. Multiple contributing factors are known to trigger the cleft formation. There are several genes involved in the aetiology of NSCLP and they are different in different populations. The genetic components of clefts that underlie the susceptibility to respond to the environment still remain unclear. In this study, five microsatellite polymorphisms from five candidate genes were employed to analyze the association between these genes and NSCLP in 83 patients and 90 controls. Genotyping was performed by separating and visualizing the fluorescently-labeled polymerase chain reaction (PCR) products. The association of the five microsatellite polymorphisms with NSCLP was tested by using the CLUMP v1.9 program that uses the Monte Carlo method. The genotypic distribution is in Hardy-Weinberg equilibrium in the control group for only the MSX1 and DLX3 genes. The RARA microsatellite was significantly associated with NSCLP. Our results suggest that the RARA gene is involved in pathogenesis of cleft lip and palate in South Indians.

Regulation and function of immunosuppressive molecule human leukocyte antigen G5 in human bone tissue

Bone-marrow mesenchymal stem cells (MSCs) are the origin of bone-forming cells with immunomodulation potential. HLA-G5 is among the generated immunosuppressive molecules. HLA-G proteins play a crucial role in promoting the acceptance of allografts. However, the mechanisms regulating the expression of HLA-G5 in human MSCs are unknown. We induced differentiation of MSCs and found that HLA-G5 was greatly up-regulated only in osteoblastic cells (+63% for mRNA). Growth plates and bone callus postfracture in adults showed that only bone-lining cells and mesenchymal progenitors were positive for HLA-G5. Use of gene silencing and dominant-negative factors revealed that HLA-G5 depends on the expression and function of the skeletogenesis master genes RUNX2 and DLX5. In addition, HLA-G5 could directly inhibit osteoclastogenesis by acting on monocytes through SHP1. However, in mature osteoblasts, the expression of HLA-G5 protein was greatly suppressed whereas the proosteoclastogenic factor, RANKL, was concomitantly increased. Down-regulation of HLA-G5 expression during the maturation of osteoblasts was due to binding of the repressor GLI3, a signal transducer of the Hedgehog pathway, to the GLI binding element within the HLA-G promoter. Our findings show that mesenchymal progenitors and osteoblastic cells specifically express HLA-G5 during osteogenesis, with a key role in bone homeostasis.

Effects of high hydrostatic pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos

Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.

Craniofacial variations in the tricho‐dento‐osseous syndrome

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant trait characterized by curly kinky hair at birth, enamel hypoplasia, taurodontism, thickening of cortical bones and variable expression of craniofacial morphology. Genetic studies have identified a 4-bp deletion in the DLX3 gene that is associated with TDO; however, phenotypic characterization and classification of TDO remains unclear in the literature. This study compares the craniofacial variations between 53 TDO-affected subjects and 34 unaffected family members. Standardized cephalograms were obtained and digitized. Cephalometric measurements were analyzed using a general linear model with family as a random effect. Numerous craniofacial measurements from both groups showed marked variability. TDO-affected subjects showed a Class III skeletal pattern (smaller SNA and ANB angles), longer mandibular corpus length (GoGn) and shorter ramus height (p < 0.05).

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell-cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages.

Novel alterations in the epigenetic signature of MeCP2-targeted promoters in lymphocytes of Rett syndrome patients

Rett syndrome (RTT) is a neurodevelopmental disorder with neurological symptoms, such as motor disorders and mental retardation. In most cases, RTT is caused by mutations in the DNA binding protein MeCP2. In mice, MeCP2 gene deletion has been reported to result in genome-wide increased histone acetylation. Transcriptional regulation of neurotrophic factor BDNF and transcription factor DLX5, essential for proper neurogenesis, is further altered in MeCP2-deleted animals. We therefore investigated the chromatin environment of MeCP2 target genes BDNF and DLX5 in lymphocytes from RTT patients and human controls, and analyzed the density of histones H3, H2B and H1, as well as the levels of methylation and acetylation on selected lysines of histone H3. Notably, we found a general increase in the density of histone H3 in RTT patients’ lymphocytes compared with controls, and decreased levels of trimethylation of lysine 4 on histone H3 (H3K4me3), a modification associated with transcriptional activation. The levels of acetylation of lysine 9 (H3K9ac) and 27 (H3K27ac) did not show any statistically significant changes when normalized to the decreased histone H3 levels; nevertheless, an average decrease in acetylation was noted. Our results reveal an unexpected alteration of the chromatin state of established MeCP2 target genes in lymphocytes of human subjects with RTT.

Developmental evidence for serial homology of the vertebrate jaw and gill arch skeleton

Gegenbaur’s classical hypothesis of jaw-gill arch serial homology is widely cited, but remains unsupported by either paleontological evidence (e.g. a series of fossils reflecting the stepwise transformation of a gill arch into a jaw) or developmental genetic data (e.g. shared molecular mechanisms underlying segment identity in the mandibular, hyoid and gill arch endoskeletons). Here we show that nested expression of Dlx genes – the “Dlx code” that specifies upper and lower jaw identity in mammals and teleosts – is a primitive feature of the mandibular, hyoid and gill arches of jawed vertebrates. Using fate-mapping techniques, we demonstrate that the principal dorsal and ventral endoskeletal segments of the jaw, hyoid and gill arches of the skate Leucoraja erinacea derive from molecularly equivalent mesenchymal domains of combinatorial Dlx gene expression. Our data suggest that vertebrate jaw, hyoid and gill arch cartilages are serially homologous, and were primitively patterned dorsoventrally by a common Dlx blueprint.

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.

Non-parsimonious evolution of hagfish Dlx genes

BackgroundThe number of members of the Dlx gene family increased during the two rounds of whole-genome duplication that occurred in the common ancestor of the vertebrates. Because the Dlx genes are involved in the development of the cranial skeleton, brain, and sensory organs, their expression patterns have been analysed in various organisms in the context of evolutionary developmental biology. Six Dlx genes have been isolated in the lampreys, a group of living jawless vertebrates (cyclostomes), and their expression patterns analysed. However, little is known about the Dlx genes in the hagfish, the other cyclostome group, mainly because the embryological analysis of this animal is difficult.ResultsTo identify the hagfish Dlx genes and describe their expression patterns, we cloned the cDNA from embryos of the Japanese inshore hagfish Eptatretus burgeri. Our results show that the hagfish has at least six Dlx genes and one pseudogene. In a phylogenetic analysis, the hagfish Dlx genes and those of the lampreys tended to be excluded from the clade of the gnathostome Dlx genes. In several cases, the lamprey Dlx genes clustered with the clade consisting of two hagfish genes, suggesting that independent gene duplications have occurred in the hagfish lineage. Analysis of the expression of these genes showed distinctive overlapping expression patterns in the cranial mesenchymal cells and the inner ear.ConclusionsIndependent duplication, pseudogenization, and loss of the Dlx genes probably occurred in the hagfish lineage after its split from the other vertebrate lineages. This pattern is reminiscent of the non-parsimonious evolution of its morphological traits, including its inner ear and vertebrae, which indicate that this group is an early-branching lineage that diverged before those characters evolved.

Developmentally Conserved Brain Designs Quantifiable with Transcriptome Tomography

Event Abstract Back to Event Developmentally Conserved Brain Designs Quantifiable with Transcriptome Tomography Yuko Okamura-Oho1, 2*, Kazuro Shimokawa3, Satoko Takemoto1 and Hideo Yokota1 1 RIKEN Center for Advanced Photonics, Japan 2 Brain Research Network (BReNt), Japan 3 National Center for Global Health and Medicine, Japan We have invented a framework for gene expression density mapping on the whole three dimensional (3D) anatomical context, transcriptome tomography, in which tissue sections in each of three orthogonal planes are fractionated (fractions), and gene expression densities in them measured with microarrays (fraction data) are then reconstructed to generate 3D maps (PLoS One 2012; 7, e45373). The fractions are exactly in the same thickness, so the fraction number of a coronal section, for instance, represents the distance from the starting point of the sections, the spino-cerebral (SC) border. Thus, besides 3D reconstructed maps, comprehensive expression densities along the body axes are measurable in the fractions. This unique feature of the data has encouraged us to test how gene expression gradients along the anterior-posterior (AP) body axis can be measured in the framework. Homeobox gene family members, Hox genes, are sequentially activated in time and space in a way of evolutionally conserved co-linear genomic position of the paralog genes (Figure 1A) and are participated in definitions of positional information along the AP axis in developmental stages of wide ranges of animals including mammals. Using the fraction data, we would show that the spatially co-linear expression patterns of Hox genes along the AP axis were also observed in the adult mouse brain. Intriguingly, the expression densities exponentially declined when plotted against the fraction numbers representing distances of from the SC border (Figure 1B), and the inclination was correlated to the genomic location of the paralogs. We would also show co-linear density gradients of Dlx genes that belonged to another homeobox gene family. The 3D expression maps of Hox and Dlx genes in the adult brain were seen in the ViBrism-DB for the first dataset created with transcriptome tomography ( http://vibrism.riken.jp/3dviewer/ex/index.html ; some seen in Figure 1C). Quantified expression density data exclusively produced with our framework provided evidences of the direct co-relation of them to the genomic organization. This may contribute to clarify a developmentally conserved regulatory mechanism underlying complex brain function. Figure 1 Acknowledgements This research was supported by Strategic Programs for R&D (President’s Discretionary Fund) RIKEN to Y.O., K.S. H.Y. et al. Keywords: digital atlasing, Gene Expression Profiling, Hox genes, Dlx genes, genomic organisation, Transcriptome Tomography Conference: Neuroinformatics 2013, Stockholm, Sweden, 27 Aug - 29 Aug, 2013. Presentation Type: Poster Topic: Digital atlasing Citation: Okamura-Oho Y, Shimokawa K, Takemoto S and Yokota H (2013). Developmentally Conserved Brain Designs Quantifiable with Transcriptome Tomography. Front. Neuroinform. Conference Abstract: Neuroinformatics 2013. doi: 10.3389/conf.fninf.2013.09.00023 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Apr 2013; Published Online: 11 Jul 2013. * Correspondence: Dr. Yuko Okamura-Oho, RIKEN Center for Advanced Photonics, Wako-Shi, Japan, yoho-tky@umin.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yuko Okamura-Oho Kazuro Shimokawa Satoko Takemoto Hideo Yokota Google Yuko Okamura-Oho Kazuro Shimokawa Satoko Takemoto Hideo Yokota Google Scholar Yuko Okamura-Oho Kazuro Shimokawa Satoko Takemoto Hideo Yokota PubMed Yuko Okamura-Oho Kazuro Shimokawa Satoko Takemoto Hideo Yokota Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Sequence analyses of the distal-less homeobox gene family in East African cichlid fishes reveal signatures of positive selection

BackgroundGen(om)e duplication events are hypothesized as key mechanisms underlying the origin of phenotypic diversity and evolutionary innovation. The diverse and species-rich lineage of teleost fishes is a renowned example of this scenario, because of the fish-specific genome duplication. Gene families, generated by this and other gene duplication events, have been previously found to play a role in the evolution and development of innovations in cichlid fishes - a prime model system to study the genetic basis of rapid speciation, adaptation and evolutionary innovation. The distal-less homeobox genes are particularly interesting candidate genes for evolutionary novelties, such as the pharyngeal jaw apparatus and the anal fin egg-spots. Here we study the dlx repertoire in 23 East African cichlid fishes to determine the rate of evolution and the signatures of selection pressure.ResultsFour intact dlx clusters were retrieved from cichlid draft genomes. Phylogenetic analyses of these eight dlx loci in ten teleost species, followed by an in-depth analysis of 23 East African cichlid species, show that there is disparity in the rates of evolution of the dlx paralogs. Dlx3a and dlx4b are the fastest evolving dlx genes, while dlx1a and dlx6a evolved more slowly. Subsequent analyses of the nonsynonymous-synonymous substitution rate ratios indicate that dlx3b, dlx4a and dlx5a evolved under purifying selection, while signs of positive selection were found for dlx1a, dlx2a, dlx3a and dlx4b.ConclusionsOur results indicate that the dlx repertoire of teleost fishes and cichlid fishes in particular, is shaped by differential selection pressures and rates of evolution after gene duplication. Although the divergence of the dlx paralogs are putative signs of new or altered functions, comparisons with available expression patterns indicate that the three dlx loci under strong purifying selection, dlx3b, dlx4a and dlx5a, are transcribed at high levels in the cichlids’ pharyngeal jaw and anal fin. The dlx paralogs emerge as excellent candidate genes for the development of evolutionary innovations in cichlids, although further functional analyses are necessary to elucidate their respective contribution.

A Novel Role of NF-YA Transcription Factor in in Vitro Adipocyte Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

AbstractAbstract 1250The CCAAT-binding transcription factor NF-Y, consists of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, however, there are no available data on the role of NF-Y in mesenchymal stem cell (MSC) biology. The aim of this study is the evaluation of NF-YA expression levels during the ex vivo expansion of human bone marrow (BM)-MSCs and the assessment of its possible role for the BM-MSC differentiation process towards the osteoblastic and adipocytic lineages.BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgery after written informed consent. BM-MSCs were ex vivo expanded until passage 10 (P10). Total RNA was isolated from BM-MSCs at P2 and P10 for the evaluation of NF-YA mRNA levels. P3 BM-MSCs’ ex vivo differentiation into adipocytes and osteoblasts was induced using the appropriate culture media. Mineralization was evidenced via Alizarin Red and Von Kosa staining and lipid droplets were revealed with Oil Red O staining. Total RNA and proteins were isolated from undifferentiated and differentiated BM-MSCs at various time-points. Protein levels of NF-YA were immunodetected by Western blot. The relative gene expression of NF-YA, as well as that of lineage-specific markers was evaluated by real-time PCR. All PCR results are expressed as 2−ΔCt. Adipocytic and osteoblastic differentiation of BM-MSCs was also induced following knockdown of NF-YA expression after BM-MSC transduction with lentiviral particles carrying either shRNA against NF-YA or nonsense shRNA. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiation.A statistically significant (p=0.04) reduction in the mRNA levels of NF-YA was found in P10, as compared to P2 BM-MSC cultures (n=12).During adipogenic differentiation, NF-YA significantly increased (p=0.0478) at day 16, as compared to the onset of differentiation induction (day 0), (n=14). Similarly, protein levels of NF-YA also increased during adipogenesis Furthermore, NF-YA expression levels strongly correlated with the mRNA levels of adipogenesis-associated genes PPARG (r=0.74, p<0.0001), C/EBPA (r=0.76, p<0.0001) and LPL (r=0.74, p<0.0001). In contrast, during osteogenic differentiation no statistically significant change was detected in NF-YA mRNA or protein expression. (n=9). In order to evaluate if NF-YA is functionally related to adipogenic and osteogenenic differentiation of BM-MSCs, NF-YA knockdown was performed and adipocytic as well as osteoblastic differentiations were induced. Throughout adipogenesis, the relative mRNA levels of NF-YA in shNF-YA-transduced BM-MSCs were reduced to 51.03% compared with control (non-transduced) cells. At day 21 of adipogenesis, a reduction of 31.22%, 70.06% and 74.3% was also observed in PPARG, C/EBPA and LPL mRNA levels, respectively. In addition, Oil Red O staining indicated a lack of lipid droplets in shNF-YA-transduced BM-MSCs compared with control cells. During osteogenesis, NF-YA mRNA levels in shNF-YA-transduced BM-MSCs were reduced to 52.37% compared to control cells. At day 21 of differentiation the mRNA levels of the osteogenesis- associated genes DLX5, RUNX2, OSX and ALP remained unaffected in shNF-YA-transduced cells compared to control cells. Furthermore, Alizarin Red and Von Kossa staining did not reveal any significant difference between shNF-YA-transduced BM-MSCs and control cells.In conclusion we have shown for the first time, that BM-MSCs display decreased NF-YA mRNA levels after prolonged ex vivo expansion, a finding that might be associated with MSCs' senescence and/or loss of stemness., We have also shown that NF-YA does not have any major effect on BM-MSCs' osteogenic differentiation but displays a significant role in their adipogenic differentiation as was shown by (a) the increased NF-YA mRNA and protein levels during the ex vivo adipogenesis, (b) the positive correlation between the NF-YA mRNA expression levels and the adipocyte-associated genes PPARG, C/EBPA and LPL and (c) the impaired adipogenic differentiation following NF-YA knock down. Collectively our results imply a novel role for NF-YA in BM-MSCs' biology Disclosures:No relevant conflicts of interest to declare.

Cleft palate defect of Dlx1/2−/− mutant mice is caused by lack of vertical outgrowth in the posterior palate

Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2-/-) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. Dlx1/2-/- mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down-regulated in the same region. Previous studies established that the epithelial-mesenchymal signaling loop involving Shh, Bmp4, and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2-/- palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2-/- mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis.

Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF, and combined VEGF plus BMP-6 in vitro

Exogenous addition of three factors—mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic proteins (BMPs)—has proven to be more beneficial than delivery of any single factor for fracture repair in animal models. We studied the osteogenic differentiation of human adipose-derived stem cells (hADSCs) in the presence of VEGF, BMP-6, or VEGF plus BMP-6 to better understand their enhancement of osteoblastic differentiation of MSCs. The VEGF plus BMP-6 group demonstrated an additive effect on the enhancement of mineralization and expression of ALP and Msx2 genes. Unlike VEGF or BMP-6 alone, the combination of VEGF and BMP-6 significantly enhanced the expression of COL1A1, osterix, and Dlx5 genes. The data indicate that a cross-talk between VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of hADSCs.

The Titanium Surface Modulates the Expression of Beta-Catenin and DLX5 Genes in Pancreatic Ductal Carcinoma in Vitro. Can the Metallic Stent Increase PDAC Aggressiveness?

Context Adenoductal pancreas (PDAC) is a fatal cancer. Its aggressiveness is associated in part with the EMT process of metastasis. Two genes specifically involved in these phenomena are b-catenin and DLX5. While the first gene has been widely studied also in pancreatic cancer, few data are associated with DLX5. However, its over-expression has been recently associated with the formation of metastases in breast cancer in vivo . An exogenous factor involved in the modulation of the expression of these genes seems to be titanium. This compound is usually employed for the pallia action of patients with PDAC, to reduce choledochal stenosis due to compression. Objective The purpose of this study was to assess whether titanium is able to modulate the expression of these two genes in vitro . Methods We used a primary cell culture of PADC (PP78). The cells were seeded and cultivated in contact with two different titanium surfaces for 10 days. After this period the total mRNA was extracted and the quantification of β-catenin and DLX5 genes was performed by RT-PCR according to the ΔΔCt analysis. Then cells were stained using the immunofluorescence technique (IF) to quantify the b-catenin protein expression .using a computerized high-resolution acquisition system (D-Sight, Menarini, Florence, Italy) The cells were scored evaluating the cytoplasmic positivity as follows (0 absent, 1 low, 2 middle, 3 strong). The experiment was carried out in triplicate and untreated cells (without titanium contact) were used as control. Results Quantitative analyses showed that both titanium surfaces positively affected beta-catenin (mean 2.8 fold) and DLX5 (2.0 fold) mRNA expressions with respect to the controls (P<0.0007). Both titanium surfaces also increased the protein score 3 values of β-catenin in treated cells with respect to their controls (P=0.0158). Conclusion Our data showed that several titanium surfaces positively modulated the expression of two genes associated with the increase of the aggressiveness of PDAC in vitro. Clinical studies are needed to find out which type of stent can be used in the surgical operation with palliative intent.

Theria‐Specific Homeodomain andcis‐Regulatory Element Evolution of theDlx3–4Bigene Cluster in 12 Different Mammalian Species

The mammalian Dlx3 and Dlx4 genes are configured as a bigene cluster, and their respective expression patterns are controlled temporally and spatially by cis-elements that largely reside within the intergenic region of the cluster. Previous work revealed that there are conspicuously conserved elements within the intergenic region of the Dlx3-4 bigene clusters of mouse and human. In this paper we have extended these analyses to include 12 additional mammalian taxa (including a marsupial and a monotreme) in order to better define the nature and molecular evolutionary trends of the coding and non-coding functional elements among morphologically divergent mammals. Dlx3-4 regions were fully sequenced from 12 divergent taxa of interest. We identified three theria-specific amino acid replacements in homeodomain of Dlx4 gene that functions in placenta. Sequence analyses of constrained nucleotide sites in the intergenic non-coding region showed that many of the intergenic conserved elements are highly conserved and have evolved slowly within the mammals. In contrast, a branchial arch/craniofacial enhancer I37-2 exhibited accelerated evolution at the branch between the monotreme and therian common ancestor despite being highly conserved among therian species. Functional analysis of I37-2 in transgenic mice has shown that the equivalent region of the platypus fails to drive transcriptional activity in branchial arches. These observations, taken together with our molecular evolutionary data, suggest that theria-specific episodic changes in the I37-2 element may have contributed to craniofacial innovation at the base of the mammalian lineage.

Distal-less 3 haploinsufficiency results in elevated placental oxidative stress and altered fetal growth kinetics in the mouse

Distal-less 3 (Dlx3)−/− mice die at E9.5 presumably due to an abnormal placental phenotype including reduced placental vasculature and secretion of placental growth factor. To examine the role of Dlx3 specifically within the epiblast, Dlx3 conditional knockout mice were generated using an epiblast-specific Meox2CreSor allele. Dlx3−/fl, Meox2CreSor animals were born at expected frequencies and survived to weaning providing indirect evidence that loss of Dlx3 within the trophoectoderm plays a critical role in fetal survival in the Dlx3−/− mouse. We next examined the hypothesis that loss of a single Dlx3 allele would have a negative impact on placental and fetal fitness. Dlx3+/− mice displayed reduced fetal growth beginning at E12.5 compared with Dlx3+/+ controls. Altered fetal growth trajectory occurred coincident with elevated oxidative stress and apoptosis within Dlx3+/− placentas. Oral supplementation with the superoxide dismutase mimetic, Tempol, rescued the fetal growth and placental cell death phenotypes in Dlx3+/− mice. To determine the potential mechanisms associated with elevated oxidative stress on the Dlx3+/− placentas, we next examined vascular characteristics within the feto-placental unit. Studies revealed reduced maternal spiral artery luminal area in the Dlx3+/− mice receiving water; Dlx3+/− mice receiving Tempol displayed maternal spiral artery luminal area similar to control Dlx3+/+ mice. We conclude that reduced Dlx3 gene dose results in diminished fetal fitness associated with elevated placental cell oxidative stress and apoptosis coincident with altered vascular remodeling. Administration of antioxidant therapy ameliorated this feto-placental phenotype, suggesting that Dlx3 may be required for adaptation to oxidative stresses within the intrauterine environment.