Dlx2 over-expression regulates cell adhesion and mesenchymal condensation in ectomesenchyme

AbstractThe Dlx family of homeodomain transcription factors have diverse roles in development including craniofacial morphogenesis and consists of 6 members with overlapping expression patterns. Dlx2 is expressed within the developing branchial arches in both the epithelium and mesenchyme and targeted deletion in mice has revealed roles in patterning and development of the craniofacial skeleton. Defects in Dlx2 null mice include skeletal anomalies of proximal branchial arch 1 derivatives while distal elements are largely spared indicating redundancy within the Dlx family. We have investigated the function of Dlx2 using in ovo electroporation and cell culture. Ectopic expression of Dlx2 within the neural tube beginning prior to emigration of neural crest cells at E1.25 drastically inhibits the migration of transfected cells and induces aggregation of transfected neuroepithelial cells within the neural tube at 24 h post-electroporation. By 48 h post-electroporation, the majority of transfected cells formed multicellular aggregates that were found adjacent to the basal side of the neural tube and very few Dlx2 expressing cells migrated to the level of the branchial arches. Similar results were obtained for Dlx5, suggesting these effects may be common to Dlx genes. Electroporation of the Dlx2 expression construct into branchial arch mesenchyme induced N-cadherin and NCAM, a dramatic increase in cell–cell adhesion relative to controls, and resulted in an increase in mesenchymal condensation. These results suggest a role for Dlx genes in regulating ectomesenchymal cell adhesion and supports the possibility that the skeletal dysmorphology seen in Dlx null mice may derive from abnormalities at the condensation stage

Global comparative transcriptome analysis of cartilage formation in vivo

<p>Abstract</p> <p>Background</p> <p>During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of <it>in vivo </it>gene expression.</p> <p>Results</p> <p>Tibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for <it>Agc1</it>, <it>Bmp8a</it>, <it>Col2a1</it>, <it>Fgfr4</it>, <it>Foxa3</it>, <it>Gdf5</it>, <it>Klf2</it>, <it>Klf4</it>, <it>Lepre1</it>, <it>Ncad</it>, <it>Sox11</it>, and <it>Trpv4</it>. Further, independent validation of the microarray data was achieved by <it>in situ </it>hybridization to analyse the expression of <it>Lepre1</it>, <it>Pcdh8</it>, <it>Sox11</it>, and <it>Trpv4 </it>from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several <it>Bmp, Gdf, Wnt, Sox and Fox </it>family members.</p> <p>Conclusion</p> <p>These data represent the first global gene expression profiling analysis of chondrogenic tissues during <it>in vivo </it>development. They identify genes for further study on their functional roles in chondrogenesis, and provide a comprehensive and important resource for future studies on cartilage development and disease.</p

Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling jeune syndrome

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies

Genome-Wide ENU Mutagenesis in Combination with High Density SNP Analysis and Exome Sequencing Provides Rapid Identification of Novel Mouse Models of Developmental Disease

BACKGROUND Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.This work was enabled by the Australian Phenomics Network and partly supported by funding from the Australian Government’s National Collaborative Research Infrastructure Strategy, a Strategic Grant from the Faculty of Medicine, Nursing and Health Sciences at Monash University, and the Victorian Government’s Operational Infrastructure Support Program. IS acknowledges support through the NH&MRC R. Douglas Wright and ARC Future Fellowship schemes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Best practices for the diagnosis and evaluation of infants with robin sequence:a clinical consensus report

Importance: Robin sequence (RS) is a congenital condition characterized by micrognathia, glossoptosis, and upper airway obstruction. Currently, no consensus exists regarding the diagnosis and evaluation of children with RS. An international, multidisciplinary consensus group was formed to begin to overcome this limitation. Objective: To report a consensus-derived set of best practices for the diagnosis and evaluation of infants with RS as a starting point for defining standards and management. Evidence Review: Based on a literature review and expert opinion, a clinical consensus report was generated. Findings: Because RS can occur as an isolated condition or as part of a syndrome or multiple-anomaly disorder, the diagnostic process for each newborn may differ. Micrognathia is hypothesized as the initiating event, but the diagnosis of micrognathia is subjective. Glossoptosis and upper airway compromise complete the primary characteristics of RS. It can be difficult to judge the severity of tongue base airway obstruction, and the possibility of multilevel obstruction exists. The initial assessment of the clinical features and severity of respiratory distress is important and has practical implications. Signs of upper airway obstruction can be intermittent and are more likely to be present when the infant is asleep. Therefore, sleep studies are recommended. Feeding problems are common and may be exacerbated by the presence of a cleft palate. The clinical features and their severity can vary widely and ultimately dictate the required investigations and treatments. Conclusions and Relevance: Agreed-on recommendations for the initial evaluation of RS and clinical descriptors are provided in this consensus report. Researchers and clinicians will ideally use uniform definitions and comparable assessments. Prospective studies and the standard application of validated assessments are needed to build an evidence base guiding standards of care for infants and children with RS

Temporal restriction of migratory and lineage potential in rhombomere 1 and 2 neural crest

AbstractMigratory cranial neural crest cells differentiate into a wide range of cell types, such as ectomesenchymal tissue (bone and connective tissues) ventrally in the branchial arches and neural tissue (neurons and glia) dorsally. We investigated spatial and temporal changes of migration and differentiation potential in neural crest populations derived from caudal midbrain and rhombomeres 1 and 2 by back-transplanting cells destined for the first branchial arch and trigeminal ganglion from HH8–HH19 quail into HH7–HH11 chicks. Branchial arch cells differentiated down ectomesenchymal lineages but largely lost both the ability to localize to the trigeminal position and neurogenic differentiation capacity by HH12–HH13, even before the arch is visible, and lost long distance migratory ability around HH17. In contrast, neural crest-derived cells from trigeminal ganglia lost ectomesechymal differentiation potential by HH17. Despite this, they retain the ability to migrate into the branchial arches until at least HH19. However, many of the neural crest-derived trigeminal ganglia cells in the branchial arch localized to the non-neural crest core of the arch from HH13 and older donors. These results suggest that long distance migration ability, finer scale localization, and lineage restriction may not be coordinately regulated in the cranial neural crest population

Detection of an appropriate kinase activity in branchial arches I and II that coincides with peak expression of the Treacher Collins syndrome gene product, treacle

Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder involving the mid and lower face and, in particular, the tissues affected arise solely from embryonic branchial arches I and II. TCOF1, the gene involved in TCS, has been cloned and although the function of the encoded protein, treacle, has not yet been established, it exhibits peak expression in the branchial arches. Treacle contains a series of repeating units of acidic and basic residues, which are predicted to contain putative casein kinase II (CKII) and protein kinase C (PKC) phosphorylation site motifs. In addition, treacle has weak homology to two phosphorylation-dependent nucleolar proteins, which shuttle between the cytoplasm and nucleolus. Based on these observations, phosphorylation of treacle may be important for its function. In this study, GST-treacle fusion peptides were constructed using particular TCOF1 exons that contained potential CKII and PKC phosphorylation sites. These were used as substrates in in vitro kinase assays and showed that treacle fusion peptides can be phosphorylated by the appropriate kinases. Furthermore, using tissue extracts we have demonstrated that in avian embryonic branchial arches I and II there is a kinase activity that can phosphorylate treacle peptides that is consistent with CKII site recognition. This activity coincides with the reported high expression of treacle in these tissues at early developmental stages and declines later in development.</p