Ethan A. Carver

The Mouse Snail Gene Encodes a Key Regulator of the Epithelial-Mesenchymal Transition

ABSTRACT Snail family genes encode DNA binding zinc finger proteins that act as transcriptional repressors. Mouse embryos deficient for the Snail (Sna) gene exhibit defects in the formation of the mesoderm germ layer. In Sna −/− mutant embryos, a mesoderm layer forms and mesodermal marker genes are induced but the mutant mesoderm is morphologically abnormal. Lacunae form within the mesoderm layer of the mutant embryos, and cells lining these lacunae retain epithelial characteristics. These cells resemble a columnar epithelium and have apical-basal polarity, with microvilli along the apical surface and intercellular electron-dense adhesive junctions that resemble adherens junctions. E-cadherin expression is retained in the mesoderm of the Sna −/− embryos. These defects are strikingly similar to the gastrulation defects observed insnail-deficient Drosophila embryos, suggesting that the mechanism of repression of E-cadherin transcription by Snail family proteins may have been present in the metazoan ancestor of the arthropod and mammalian lineages.

Developmental transcription factor Slug is required for effective re-epithelialization by adult keratinocytes

During re‐epithelialization of cutaneous wounds, keratinocytes recapitulate several aspects of the embryonic process of epithelial‐mesenchymal transition (EMT), including migratory activity and reduced intercellular adhesion. The transcription factor Slug modulates EMT in the embryo and controls desmosome number in adult epithelial cells, therefore, we investigated Slug expression and function during cutaneous wound re‐epithelialization. Slug expression was elevated in keratinocytes bordering cutaneous wounds in mice in vivo, in keratinocytes migrating from mouse skin explants ex vivo, and in human keratinocytes at wound margins in vitro. Expression of the related transcription factor Snail was not significantly modulated in keratinocytes during re‐epithelialization in vitro. Epithelial cell outgrowth from skin explants of Slug knockout mice was severely compromised, indicating a critical role for Slug in epithelial keratinocyte migration. Overexpression of Slug in cultured human keratinocytes caused increased cell spreading and desmosomal disruption, both of which were most pronounced at wound margins. Furthermore, in vitro wound healing was markedly accelerated in keratinocytes that ectopically expressed Slug. Taken together, these findings suggest that Slug plays an important role during wound re‐epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development.

Slug Is a Novel Downstream Target of MyoD TEMPORAL PROFILING IN MUSCLE REGENERATION

Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression, promoter data base mining, and gel shift assays; Slug and calpain 6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown to be a downstream target by using promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice.

Craniosynostosis in Twist heterozygous mice: a model for Saethre-Chotzen syndrome.

Saethre‐Chotzen syndrome is a common autosomal dominant form of craniosynostosis, the premature fusion of the sutures of the calvarial bones of the skull. Most Saethre‐Chotzen syndrome cases are caused by haploinsufficiency for the TWIST gene. Mice heterozygous for a null mutation of the Twist gene replicate certain features of Saethre‐Chotzen syndrome, but have not been reported to exhibit craniosynostosis. We demonstrate that Twist heterozygous mice exhibit fusions of the coronal suture and other cranial suture abnormalities, indicating that Twist heterozygous mice constitute a better animal model for Saethre‐Chotzen syndrome than was previously appreciated. Anat Rec 268:90–92, 2002.

LOCATION OF MOUSE AND HUMAN GENES CORRESPONDING TO CONSERVED CANINE OLFACTORY RECEPTOR GENE SUBFAMILIES

Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved olfactory receptor genes, each representing separate, recently identified canine gene subfamilies. Three of the four canine genes detected related gene clusters in regions of mouse Chromosomes (Chrs) 2, 9, and 10, near previously mapped mouse olfactory genes, while one detected a formerly unidentified gene cluster located on mouse Chr 6. In addition, we have localized two human gene clusters with homology to the canine gene, CfOLF4, within the established physical map of Chr 19p. Combined with recently published studies, these data link the four conserved olfactory gene subfamilies to homologous regions of the human, dog, and mouse genomes.

A reciprocal translocation dissects roles of Pax6 alternative promoters and upstream regulatory elements in the development of pancreas, brain, and eye

Pax6 encodes a transcription factor with key roles in the development of the pancreas, central nervous system, and eye. Gene expression is orchestrated by several alternative promoters and enhancer elements that are distributed over several hundred kilobases. Here, we describe a reciprocal translocation, called 1Gso, which disrupts the integrity of transcripts arising from the 5′‐most promoter, P0, and separates downstream promoters from enhancers active in pancreas and eye. Despite this fact, 1Gso animals exhibit none of the dominant Pax6 phenotypes, and the translocation complements recessive brain and craniofacial phenotypes. However, 1Gso fails to complement Pax6 recessive effects in lacrimal gland, conjunctiva, lens, and pancreas. The 1Gso animals also express a corneal phenotype that is related to but distinct from that expressed by Pax6 null mutants, and an abnormal density and organization of retinal ganglion cell axons; these phenotypes may be related to a modest upregulation of Pax6 expression from downstream promoters that we observed during development. Our investigation maps the activities of Pax6 alternative promoters including a novel one in developing tissues, confirms the phenotypic consequences of upstream enhancer disruption, and limits the likely effects of the P0 transcript null mutation to recessive abnormalities in the pancreas and specific structures of the eye. genesis 51:630–646.

Physical mapping of EMR1 and CD97 in human Chromosome 19 and assignment of Cd97 to mouse Chromosome 8 suggest an ancient genomic duplication.

EMR1 and CD97 belong to the EGF-TM7 family of nonclassicalseven-span transmembrane receptors, members of which are ex-pressed primarily in the immune system. The membrane-spanningregions of CD97, EMR1, and other EGF-TM7 proteins show sig-nificant homology to the secretin receptor superfamily. However,unlike this group of peptide hormone receptors, EMR1 and CD97have extended extracellular portions that possess several EGF do-mains at the N-terminus (McKnight and Gordon 1998). The EGFdomain region can function as a ligand-binding site, as demon-strated by the interaction of CD97 with CD55 (Hamann et al.1996). EMR1 and CD97 show a high degree of structural similar-ity despite the fact that they share only about 31% amino acidsequence identity. The similarity between these two proteins sug-gests that CD97 and EMR1 coding sequences arose through an-cient duplication of a common ancestral gene. CD97 has beenassigned to human Chromosome (Chr) 19p13.12–p13.2 by fluo-rescence in situ hybridization (FISH; Hamann et al. 1995), andEMR1 has been mapped to Chr 19p13.3 through a combination ofFISH and somatic cell hybrid analysis (Baud et al. 1995). Thesedata indicate that the two related genes are linked but separated bya significant distance in the human genome.Emr1 has been mapped to distal mouse Chr 17 within a regionrelated to human 19p13.3, tightly linked to the gene encodingtranscription factor Rfx2 (Lin et al. 1997; McKnight et al. 1997).Interestingly, RFX1, which encodes a transcription factor proteinrelated to RFX2 in both structure and immune-system function,has been mapped to 19p13.1 (Doyle et al. 1996), suggesting thathuman RFX1 and CD97 might also be close neighbors. RFX1 andRFX2 encode site-specific DNA binding proteins that serve criti-cal immune-system functions (Reith et al. 1994) and are alsothought to have arisen from a common ancestral gene sequence indistant evolutionary time. These data suggested that the tight link-age of RFX2 to EMR1, and RFX1 to CD97, respectively, mightreflect an ancient duplication encompassing the predecessors ofboth sets of immunologically active genes.To investigate this possibility, we set out to define physicallocations of CD97 and EMR1 genes in the human and to determinethe location of Cd97 in mice. We localized the mouse Cd97 geneby following the segregation of variant M. musculus and M. spre-tus alleles of the gene in an interspecific backcross (Doyle et al.1996; Stubbs et al. 1996). The results confirmed the tight linkageof Cd97 and Rfx1 in central mouse Chr 8 (Fig. 1). To define thepositions of human CD97 and EMR1, we hybridized probes rep-resenting the two genes to a Chr 19 cosmid library (Olsen et al.1994), and positive cosmids were ordered within the Chr 19 metricphysical map (Ashworth et al. 1995). The human CD97 probeidentified several overlapping cosmids located in 19p13.1 betweenthe RFX1 and NOTCH3, approximately 700 kb and 400 kb awayfrom those two genes, respectively (Fig. 2). The EMR1 probedetected two cosmid clones, 31568 and 34349; positive hybridiza-

Modifications of ribonuclease A induced by p-benzoquinone

The nature of ribonuclease A (RNase) modifications induced by p-benzoquinone (pBQ) was investigated using several analysis methods. SDS-PAGE experiments revealed that pBQ was efficient in producing oligomers and polymeric aggregates when RNase was incubated with pBQ. The fluorescence behavior and anisotropy changes of the modified RNase were monitored for a series of incubation reactions where RNase (0.050 mM) was incubated with pBQ (0.050, 0.25, 0.50, 1.50 mM) at 37 °C in phosphate buffer (pH 7.0, 50 mM). The modified RNase exhibited less intense fluorescence and slightly higher anisotropy than the unmodified RNase. UV-Vis spectroscopy indicated that pBQ formed covalent bonds to the modified RNase. Confocal imaging analysis confirmed the formation of the polymeric RNase aggregates with different sizes upon exposure of RNase to high concentrations of pBQ. The interaction between the modified RNase and salts affecting biomineralization of salts was also investigated by scanning electron microscopy. Overall, our results show that pBQ can induce formation of both RNase adducts and aggregates thus providing a better understanding of its biological activity.

Use of flatbed transparency scanners in zebrafish research: versatile and economical adjuncts to traditional imaging tools for the Danio rerio laboratory.

Flatbed transparency scanners are typically relegated to routine office tasks, yet they do offer a variety of potentially useful imaging tools for the zebrafish laboratory. These include motility screens, oocyte maturation and egg activation assays as well as counting and measuring tasks. When coupled with Macroscheduler (http://www.mjtnet.com) and ImageJ (http://rsbweb.nih.gov/ij), the scanner becomes a stable platform for imaging large arrays of zebrafish oocytes, embryos, larvae, and adults. Such large arrays are a prerequisite to the development of high-throughput screens for small molecules as potential therapeutic drugs in the treatment of many diseases including cancer and epilepsy. Thus the scanner may have a role in adapting zebrafish to future drug and mutagenesis screening. In this chapter, some of the uses of scanners are outlined to bring attention to the potentials of this simple-to-use, flexible, inexpensive device for the zebrafish research community.

Location of the DBP transcription factor gene in human and mouse

tBiology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, Tennessee 37831-8077, USA 2University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831-8077, USA 3Human Genome Center, Lawrence Livermore National Laboratory, P.O. Box 808, L-452, Livermore, California 94551, USA 4Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland