POSTER 57 - IMPROVING AXIS DEFECTS: A SUPPRESSOR OF BRACHYURY (SuB) ON MOUSE CHROMOSOME 6

Prof Karen Artzt
University of Texas at Austin
Institute for Cellular and Molecular Biology
Section of Molecular Genetics and Microbiology; A4800
Austin
78712-1064
USA
Artzt@uts.cc.utexas.edu

Co-Authors: Wu J, Centilli M, Vasquez G, Young S, Scolnick J, Spearow JL

Institution:  University of Texas at Austin Institute for Cellular and Molecular Biology, University of California at Davis

Improving axis defects: A suppressor of brachyury (SuB) on mouse chromosome 6

During a microsatellite genome scan to map tint (T/t tail interaction factor), we discovered a new hypomorphic gene that suppresses the Brachyury (T)phenotype.  Homozygotes for the T/T die of serious axis defects on E10.5 while heterozygotes have a short tail.  Double heterozygosity for both T and SuB have normal tails. To our knowledge, this is the first gene that improves axis defects and therefore may be relevant to spina bifida.

The origin for Sub is a spretus subline that was used in our intercross (Spr/Jls-RP):  F1 mice were wild type at the T locus and heterozygous for tint versus spretus. Since all the F1s were normal tailed, to monitor the transmission of tint, it was necessary to testcross to T/+, +/+.  We expected 1/2 normal tails, 1/4 shorts (T/+, spretus/+) and 1/4 tailless (T/+, tint/+) offspring.  The normal tails would be of two indistinguishable genotypes and were discarded.  In 713 testcross progeny, instead of the expected 2:1:1 ratio, we found a 2.5:1:0.5 ratio.  When markers on chromosome 6 were typed, virtually all the tailless mice were of the tint type.  The exceptions were recombinants for neighboring markers.  The data map SuB to spretus chromosome 6 at 35.22 cM inseparable from D6Mit22 but distinct from D6Mit386.  When T/+ was crossed to the input spretus stock, in the absence of any interaction, half the progeny are expected to be short tailed. The fact that 59/59 progeny had normal tails argues that we identified a suppressor of T.

  POSTER 58 - TRANSCRIPT MAP OF THE Om LOCUS

Dr Patricia Baldacci
Institut Pasteur
Unité Biologie du Développement
Institut Pasteur, 25 Rue de Dr Roux
Paris
75724 Paris Cedex 15
France

Co-Authors:  Le Bras S, Cohen-Tannoudji M, Guyot V, Vandormael-Purnin S, Couailleau F, Babinet C

Institution:  Unité Biologie du Développement, Institut Pasteur

Transcript map of the Om locus

The DDK syndrome is defined as the embryonic lethality, at the morula-blastocyst stage, of F1 embryos from crosses between DDK females and males from other strains. Genetically controlled by the Om locus, it is due to a deleterious interaction between a maternal factor present in DDK oocytes and the non-DDK paternal pronucleus. Thus, the DDK syndrome constitutes a unique genetic tool to study the crucial interactions that take place between the parental genomes and the egg cytoplasm during mammalian development. We have performed an extensive analysis of the Om region by exon trapping. 150 unique sequences were identified of which 27 corresponded to genes in the databases: b-adaptin, CCT zeta2, DNA LigaseIII, Notchless, Rad51l3 and Scya1. 28 other sequences presented homologies with ESTs and genomic sequences whereas 57 did not. The pattern of expression of 37 of these markers was established by RT-PCR. Importantly, the expression of 5 sequences was detected in DDK oocytes making them candidate genes for the maternal factor. 20 sequences were expressed in testis and are candidate genes for the paternal factor. These results are an important step forward in identifying the genes responsible for the DDK syndrome.

POSTER 59 - A GENOMIC APPROACH TO ANALYZE GENES INVOLVED IN NERUAL CREST-MELANOCYTE DEVELOPMENT

Dr Laura Baxter
NIH/NHGRI
Bldg 49, Rm 4A67
49 Convent Drive
Bethesda
20892
USA

Co-Authors:  1)Loftus SK, 1)Larson DM, 1)Antonellis A, 1)Watkins-Chow D, 2)Jiang Y, 2)Chen Y, 2)Bittner M, 2)Trent J, 1)Pavan WJ

Institutions: 1)NIH/NHGRI/Genome Disease Research Branch, 2)NIH/NHGRI/Cancer Genetics Branch,

A genomic approach to analyze genes involved in neural crest-melanocyte developement

Melanocytes (MC)--pigment producing cells derived from the neural crest--are responsible for hair and skin coloration. Alterations in MC development and function result in human disorders including Waardenburg syndrome (SOX10, PAX3 and MITF), albinism (TYR, TYRP1 and DCT), and melanoma. While mutations in SOX10, PAX3 and MITF transcription factors result in MC deficiencies, few downstream targets of these genes have been identified. To identify potential targets, a 4356 human MC cDNA clone set was used for microarray expression profile analysis. Hierarchical clustering identified two gene clusters with expression patterns similar to genes with known MC function. The first 81 gene cluster (Neural Crest) contained genes previously shown to be involved in neural crest-melanocyte (NC-MC) formation, including SOX10, MITF and EDNRB. The second 72 gene cluster (Pigmentation) contained genes critical to melanin synthesis (TYRP1, CHS and DCT). Bioinformatic analysis of the cluster genes determined genomic localization, putative function, and potential association with human NC-MC disorders. Murine homologs were identified, and are being analyzed as candidates for uncloned pigmentation mutants. Developmental in situ hybridization analysis in normal and transcription factor mutant mice revealed expression patterns of these genes, thus identifying transcriptional regulatory networks for MC's and discovering novel genes involved in NC-MC development.

POSTER 60 - THE HARWELL MOUSE IMPRINTING MAP

Colin V Beechey
Mammalian Genetics Unit
Harwell
Didcot
Oxon
OX11 0RD
UK

Co-Author:  Cattanach BM

Institution:  Mammalian Genetics Unit, Harwell

The Harwell mouse imprinting map

An up-to–date version of the Harwell Mouse Imprinting Map with associated data and references is presented. The map illustrates those regions of the genome subject to imprinting as defined by the occurrence of anomalous phenotypes with uniparental duplication.  These imprinting regions, and the chromosome anomalies defining them, are illustrated on both the genetic and G-banded map with all currently known imprinted genes. Human homologous regions are also shown. Almost all identified imprinted genes map to these imprinting regions although a small number do not.  One such gene is Rasgrf1, located on distal Chr 9.  Maternal transmission of a Rasgrf1 knockout leads to postnatal growth retardation.  Re-investigation of distal Chr 9, using several different translocations to generate uniparental duplications, has now identified a similar postnatal growth retardation with maternal duplication.  This effect was previously attributed to distal Chr 17 but these new findings mean that the conflict in the data no longer exists.  Another investigation has focused on proximal Chr 2, which has shown variable evidence of imprinting. New studies have firmly established that a region close to the centromere is subject to imprinting with effects on foetal viability, growth and placental size, although to date no imprinted genes have been identified here. The imprinting regions shown on the Harwell Map now define the sites of all but 3 identified imprinted genes, Htr2a (Chr14), Ins1 (Chr 19) and Zac1 (Chr10). This inconsistency could be due to subtle or variable imprinting phenotypes associated with uniparental duplication of these chromosome regions.

POSTER 61 - GENETIC STUDY OF PREAXIAL POLYDACTYLY USING MOUSE MODELS

Dr Oh Bermseok
Korea National Institute of Health
5 Nokbun-dong, Eunpyung-gu
Seoul
122-701
Korea

Co-Authors:  Jin SJ, Koo SK, Lee KS, Park DH, Jung SC

Institution:  Korea NIH, Seoul

Genetic study of preaxial polydactyly using mouse models

Polydactyly is a congenital anomaly of digit duplication caused by the mutation of genes involved in limb development. Preaxial polydactyly, thumb duplication is the most frequent form among the polydactyly. The genetics is not completely clear though many cases of familial inheritance have been reported. To study the mechanism of polydactyly and identify the causative genes, two preaxial polydactyly mice, mes and Dh, have been investigated in the laboratory. mes has an complete penetrance of thumb biphalangeal polydactyly in an autosomal recessive manner. Differently from other polydactyly model mice mes does not show syndactyly or oligodactyly, therefore this mutant is an exellent model to study the mechanism of preaxial polydactyly. Last year the candidate gene for the mes has been reported as patched, receptor of anterior-posterior patterning gene Shh, but no ectopic expression of genes involved in the Shh-Ptc pathways was found. We hypothesize that preaxial polydactyly in mes is caused by the outgrowth of anterior region of limb bud diectely or indirectly by the truncated Ptc. To prove it we study the proliferation and apoptosis of the anterior region in mes and also examine any change in genes involved in the Shh-Ptc pathway. Dh also has polydactyly. mostly the preaxial type of triphalanx, oligodactyly or syndactyly in addition to tibial hemimelia in an autosomal dominant way. We are cloning the gene by positional cloning.

POSTER 62 - THE ROLE OF BARX-1 IN TOOTH DEVELOPMENT

Mr Georg Buechner
Department of Craniofacial Development
Guy's tower 28th Floor
Guy's hospital
London
SE1 9RT

Co-Author:  Sharpe PT

Institution:  Guy's hospital

The role of barx-1 in tooth development

Current research in Prof. Sharpe's lab is focused on understanding the molecular control of odontogenesis Prof. Sharpe has proposed the so-called homeobox-code model to explain the segmental patterning and shape differences between the different types of tooth.  In this model it is proposed that tooth shape and position are specified by the combinatorial action of different homeobox genes expressed in neural crest derived facial mesenchyme. My project is to study the effects of manipulating the expression of one of the homeobox genes known to be involved in odontogenesis: Barx1. The proximal expression of Barx1 in the molar-specific mesenchymal regions of the first branchial arch, prior to the initiation of tooth development, seems to drive the formation of the molars.

My project will test these hypothesis by: 1Generating of  a Barx1 knock-out. I have obtained 6 positive Es clones that are currently been inject into blatocyst .2        Ectopic expression of Barx1 in the incisor region using an IRES-GFP expression vector. This project involves the electroporation of a Barx1 expression vector in the incisor region of dissected E10 mandibles. I have obtained widespread ectopic expression of Barx-1 in the mesenchyme of the incisor region. I am now culturing these explants for 14 days in mouse renal capsule for tooth development to progress  so that any change of incisor identity can be studied. I am also performing a molecular analysis using key genes known to be involved in tooth development.

POSTER 63 - DEPLETION OF A SUBSET OF SYNAPTIC VESICLES IN MICE LACKING A-SYNUCLEIN

Dr Deborah Cabin
NHGRI/NIH
49 Convent Dr.
Building 49, Room 4B67
Bethesda
20892
MD

Co-Authors:  1)Murphy D, 2)Gottschalk W, 2)Lu B, 3)Nussbaum R

Institutions:  1)NINDS/NIH, 2)NICHD/NIH, 3)NHGRI/NIH

Depletion of a subset of synaptic vesicles in mice lacking a-synuclein

Mutations in the a-synuclein gene (SNCA) are responsible for a subset of familial forms of Parkinson's disease.  The function of the SNCA protein is unknown, but it is the major component of Lewy bodies, the neuronal inclusions that are diagnostic for Parkinsons disease.  Mice homozygous for a targeted deletion in Snca produce no a-syncuclein detectable on Western blots, and are viable, healthy, and appear to have normal life spans.  As the protein is presynaptic, electron microscopy was used to examine synaptic structure in both cultured hippocampal neurons and in hippocampal sections.  Mutant animals had significantly fewer synaptic vesicles in the vesicle cluster proximal to the docked vesicle pool.  Two electrophysiological paradigms were used to determine if this difference is reflected in synaptic function.  No difference was seen between mutant and wild type mice in response to a train of high frequency stimuli sufficient to exhaust docked synaptic vesicles.  A prolonged course of low frequency stimuli in which the reserve pool of synaptic vesicles is believed to be utilized suggests that this pool is depleted more quickly in the mutant than in normal mice.  Snca thus seems to play a role in the production or maintenance of a subset of synaptic vesicles.  cDNA subtraction experiments are now underway to determine if these phenotypes are associated with changes in gene expression in the mutant mice.

POSTER 64 - GENETICS OF DEAFNESS: ROLE OF MYOSIN 15 (MYO15)

Dr Sally Camper
University of Michigan
4301 MSRB 3
1500 W. Med. Ctr. Dr.
Ann Arbor
48109-0638
USA

Co-Authors:  Karolyi IJ, and Rapael Y

Institution:  University of Michigan

Genetics of deafness: role of myosin 15 (myo15)

Shaker 2 mice have profound congenital deafness and vestibular dysfunction. We mapped sh2 on mouse Chr11 in a region homologous to human Chr 17, which implicated sh2 as a model for DFNB3. The hunt for the mutated gene was simplified by complementation with a BAC transgene. DNA sequence analysis revealed mutations in the MYO15 gene in sh2 mice and individuals with DFNB3. Myosins contain molecular motors that bind cytoskeletal actin and hydrolyze ATP to produce force and movement. The tail domains are heterogeneous and poorly understood. The sh2 allele has a missense mutation in the motor domain. The sh2J lesion is a deletion of the last six exons, which encode a FERM domain that interacts with integral membrane proteins. The FERM domain is as critical as the motor for hearing and balance. Both the sh2 and sh2J alleles result in abnormally short hair cell stereocilia, suggesting that Myo15 is important for the structure and function of these sensory epithelia. Mouse genetics identified two other myosin genes required for normal hearing in humans and mice; Myo6 and Myo7a. The long stereocilia in Myo6 mutants suggest involvement in membrane anchoring. The highly disorganized stereocilia of Myo7a mutants suggests a role in patterning stereocilia.  Double mutant phenotypes suggest each myosin has a specialized role, with few overlapping functions that are compensated for in single mutants.

POSTER 65 - THE X-LINKED MOUSE MUTATION BENT TAIL IS ASSOCIATED WITH A DELETION OF THE ZIC3 LOCUS

Ms Tessa Carrel
Children's Research Institute & Dept of Pediatrics
The Ohio State University
Columbus
OH 43221
USA

Co-Authors: 1) Purandare SM, 2) Parker M, 2) Fox T, 1) Casey B, 2) Herman GE

Institutions: 1) Dept of Pathology, Baylor College of Medicine, 2) Children’s Research Institute & Dept of Pediatrics, The Ohio State University

Bent tail (Bn) is a spontaneous X-linked mutation that produces tail deformities and, occasionally, an open neural tube.  We have demonstrated that the Bn mutation is associated with a 60-170 kb submicroscopic deletion that includes the entire Zic3 locus.  Human mutations in ZIC3 are associated with left-right axis malformations and anal defects, including NTDs. Situs anomalies were found in Bn males and females, the most frequent being partial abdominal situs ambiguus.   The laboratory of Dr. Brett Casey has constructed a mouse strain carrying a targeted null allele of Zic3, which has a strikingly similar phenotype to mice with the Bn mutation.  By analyzing 153 progeny from complementation breeding between Bn females and Zic3 deficient males, we believe that the Bn phenotype is due solely to the absence of the Zic3 gene.  Clinical studies in humans have shown that, during pregnancy, maternal diets enriched in folic acid can reduce the occurrence of most NTDs, even if the offspring are genetically predisposed.  Feeding Bn females folic acid enriched or depleted diets had no effect on the frequency or the severity of the Bn phenotype in the viable progeny.  We are currently investigating the effect on developing Bn embryos.  Finally, by breeding both Bn and Zic3-/- alleles onto different genetic backgrounds, we have been able to separate the tail and situs phenotypes.  This will allow us to map modifier loci for each phenotype individually.

POSTER 66 - AN ENHANCER ELEMENT AT THE Igf2/H19 LOCUS DRIVES GENE EXPRESSION IN BOTH IMPRINTED AND NON-IMPRINTED TISSUES

Marika Charalambous
Developmental Biology Program
School of Biology and Biochemistry
University of Bath
Claverton Down
Bath
BA2 7AY

Co-Author:  Ward A

Institution:  Developmental Biology Program, University of Bath

An enhancer element at the igf2/h19 locus drives gene expression in both imprinted and non-imprinted tissues

The Insulin-like growth factor 2 (Igf2) and H19 genes lie within 100kb of each other on mouse chromosome 7, and are thought to share cis- elements necessary for both expression and imprinting. The paternally expressed Igf2 gene encodes a fetal growth factor, and the maternally expressed H19 gene a non-coding RNA of unknown function. In the developing endodermal tissues, imprinting of these genes is thought to be mediated by the interaction of a set of enhancers downstream of the H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4kb upstream of H19. In the remainder of tissues that express Igf2 and H19, the cis- elements that drive their correct expression and imprinting are not well understood, but may rely upon the differential methylation of several sites proximal to the Igf2 gene. In addition, the enhancers driving the expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is not imprinted, have not been isolated.

Approximately 32kb upstream of the mouse H19 gene lies a region containing two blocks of strong sequence conservation between mouse and human, and that exhibits DNaseI hypersensitivity and hypomethylation relative to the surrounding. We have created transgenes containing 2kb of genomic DNA containing this region, that we have termed the centrally conserved domain (CCD), in-cis to a Igf2-promoter 3-LacZ reporter gene. Transgenic mice bearing these constructs show reporter gene expression in a variety of tissues where Igf2 is normally expressed, including the choroid plexus, leptomeninges, tongue, facial mesenchyme and lens of the eye. We propose that this intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and in regions where Igf2 is biallelically expressed. The existence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation proximal to Igf2.

POSTER 67 - PERI-IMPLANTATION LETHALITY OF MICE LACKING OVUM MUTANT CANDIDATE GENE 1 (OMCG1)

Michel Cohen-Tannoudji
Unite de Biologie du Developpement/CNRS URA 1960.
Institut Pasteur
25, rue du Docteur Roux
75724 Paris Cedex 15
France

Co-Authors:  Frödin M, Vandormael-Pournin S, Artus J, Le Bras S, Coumailleau F, Baldacci P, Bainet C

Institution:  Unite de Biologie du Developpement/CNRS URA 1960.

Peri-implantation lethality of mice lacking Ovum mutant candidate gene 1 (omcg1)

The locus Ovum mutant (Om), described in the DDK strain of mice, affects the viability of preimplantation embryos. The striking property of this locus is a parent-of-origin effect: embryos from a cross between DDK females x non-DDK males die at the morula to blastocyst stage whereas embryos from the reciprocal cross, non-DDK females x DDK males, are viable. This lethality is caused by an incompatibility, at the one-cell stage, between a maternal factor of DDK origin and a paternal component of non-DDK origin. Both maternal and paternal components segregate as a single mendelian locus, the Om locus, which we have mapped on mouse chromosome 11. We are engaged in the identification of the gene(s) responsible for the observed lethality using a positional cloning strategy. The Omcg1 gene has been isolated by cDNA selection/hybridisation of DDK oocyte cDNAs with a BAC of the Om region. The Omcg1 gene codes for a putative 40 kDa nuclear protein of unknown function. Interestingly, the Omcg1 gene is expressed in both male and female germ cells. In order to elucidate its biological function, we inactivated the Omcg1 gene by gene targeting. Omcg1-null embryos die in utero. They develop normally to the blastocyst stage but present defaults in hatching and/or implantation. Detailed analysis of the phenotype will help to refine the role of Omcg1 in early mammalian development.

POSTER 68 - Nell1: A CANDIDATE GENE FOR ENU-INDUCED RECESSIVE LETHAL MUTATIONS AT THE l7R6 LOCUS AND POTENTIAL MOUSE MODELS FOR HUMAN NEONATAL UNILATERAL CORONAL SYNOSTOSIS (UCS)

Cymbeline T Culiat
Life Sciences Division
Oak Ridge National Laboratory
P.O. Box 2009
Oak Ridge
TN 37831-8077

Co-Authors: 1)Millsaps J, 1)Desai J, 2)Stanford B, 2)Hughes L, 2)Kerley M, 2)Carpenter D, 2&3)Rinchik EM

Institutions:  1)Graduate School of Genome Science & Technology, The University of Tennessee, 2)Life Sciences Division, Oak Ridge National Laboratory, 3)Dept. of Biochemistry, Cellular & Molecular Biology, The University of Tennessee

NELL1: A candidate gene for enu-induced recessive lethal mutations at the l7r6 locus and potential mouse models for human neonatal unilateral coronal synostosis (ucs)

A gene (l7R6) critical for late embryonic development and survival has been mapped proximal to the pink-eyed dilution (p) gene in mouse chromosome 7. Six independent ENU-induced alleles designated 88SJ, 335SJ, 2038SJ, 102DSJ, 11DSJ and 45DSJ all result in late-gestation/neonatal lethality.  l7R6 maps to a region homologous to human 11p15.1, that contains a very large gene for a protein kinase C binding protein, called NELL1. Human NELL1 has a 2433-bp coding region with at least 15 exons spread out in ~500 kb genomic distance. Because the human gene is so large, and because we recovered so many l7R6 alleles in a relatively small number of gametes, the mouse counterpart seemed a logical candidate for l7R6. To determine if l7R6 is Nell1, a near full-length (1920 bp) cDNA was generated and was used as a probe for Northern analysis of both wild-type and mutant animals.  Nell1 expression was detected from E10-E18, increasing as fetal development progresses and concentrating particularly in the head at E18. In wild-type adults, expression was predominantly in brain. Notably, a severely reduced level of Nell1 expression was detected in one allele (102DSJ).  Abnormal expression of human NELL1 is associated with unilateral coronal synostosis (UCS) in newborns, a condition where coronal sutures fuse early, resulting in abnormal head development and limb defects. Mouse hemizygotes recovered at either E18 or two hours after birth also exhibit both gross cranial and limb defects. This phenotype thus support the Northern analysis result indicating that Nell1 is l7R6 and that Nell1 mutations in mouse resemble major components of NELL1 defects in humans. Cloning and sequencing of RTPCR-derived cDNA clones from mutant and wild-type alleles are in progress. [Research sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy under contract DE-AC05-00OR22725 with UT-Batelle, LLC.]

POSTER 69 - DEVELOPMENTAL EPIGENETIC CHANGES AT THE CPG ISLAND OF THE MOUSE SMCX GENE IN RELATION TO ESCAPE FROM X INACTIVATION

Dr Christine Disteche
University of Washington
BOX 357470
Department of Pathology
Seattle
WA 98195
USA

Co-Authors:  1)Thomas S, 1)Truong J-P, 2)Tsuchiya K

Institutions:  1)Department of Pathology, University of Washington, 2)Department of Pathology, Vanderbilt University 

Developmental epigenetic changes at the CPG island of the mouse SMCX gene in relation to escape from X inactivation

Smcx is a mouse gene that escapes X inactivation in adult mice.  Our previous studies have shown that Smcx can be completely inactivated in cells of early mouse embryos.  This developmental change in the regulation of Smcx on the inactive X chromosome suggests that genes that escape X inactivation may undergo a process of reactivation during development.  In the present study we examined the methylation patterns of Smcx CpG island in mouse embryos and adult tissues.  The methylation status of CpG dinucleotides within the CpG island of the Smcx gene sequence was determined using the bisulfite method.  No methylation of the CpG island was found in adult tissues, as expected for a gene that escapes X inactivation.  In contrast, our results indicate that a specific region of the CpG island of Smcx is methylated in mouse embryos.  This cluster of methylated sites may be involved in the regulation of expression of the gene on the inactive X chromosome.  Our analysis is consistent with progressive loss of DNA methylation associated with establishment of the inactivation escape in adult mice.

POSTER 70 - CHARACTERISATION OF THE KUMBA MOUSE MUTANT

Mr Paul Elms
MRC Harwell
Mammalian Genetics Unit,
Harwell
Didcot
OX11 0RD
UK

Co-Authors:  Siggers P, Haines H, Alexander E, Greenfield A, Arkell R 

Institution: Mammalians Genetics Unit, Harwell

Characterisation of the kumba mouse mutant

The Kumba (Ku) mouse mutant was recovered from a genetic screen for dominant, ENU-induced phenotypes.

On some genetic backgrounds, Ku/+ mice exhibit a looped tail, which is occasionally accompanied by spina bifida, making the Kumba mutant a model for neural tube disorders.  Additionally, many Ku/+ mice have a ventral spot and some have a limb defect.  On the C3H background, the mutation shows incomplete penetrance, with one third of heterozygotes appearing normal.  Mice that are homozygous for the mutation die during mid-gestation.

To further investigate the developmental defect associated with this mutation we are characterising the homozygous phenotype at 9.5 dpc.  At this stage, visual analysis of the Ku/Ku embryos reveals delayed neural tube closure, incomplete embryonic turning, incorrect heart looping, a deformed forebrain and a lack of 2nd branchial arch formation.  Analysis with molecular markers has revealed additional defects including aberrant hindbrain segmentation. 

The Ku mutation is linked to distal Chromosome14.  We have shown that the phenotype is caused by a mutation in a zinc finger transcription factor which is expressed throughout the developing nervous system and in the somites, limb buds and eye.

POSTER 71 - A MOUSE TRANSLOCATION CAUSING SEVERE GLOMERULONEPHRITIS

Ms. Mary Guarnieri
Wadsworth Center
P.O. Box 22002
Albany, NY
12201
USA

Co-Authors: 1) Cacheiro, NLA,    2) Rudofsky, UH, 2) Collins, DN, 2) Flaherty, LA

Institutions: 1) Life Sciences Division, Oak Ridge National Laboratory, 2) Wadsworth Center,

A chlorambucil-induced t(2;13) translocation causes a series of abnormalities including open eyelids at birth, coat color dilution, and neurological problems.  Mice bearing this translocation die before 3 months of age.  Our recent results have shown that this death is most likely due to the rapid development of glomerulonephritis. At 1-6 months, mutant mice show high blood urea nitrogen levels and develop severe proteinuria. Immunofluorescence of mutant kidneys showed granular staining of IgG, IgM and C3 in glomerular loops and mesangial cells. Additionally, double-stranded DNA auto-antibodies have been found in sera from mutant mice. The above data indicate the presence of an autoimmune disorder in these mice that resembles systemic lupus erythematosus (SLE).

Using fluorescent in-situ hybridization (FISH), we have mapped the position of this mutation/translocation more precisely. A contig spanning the breakpoint region on chromosome 13 was assembled with the RPCI-23 BAC library. These BACs were hybridized to mutant metaphase chromosomes.  Two overlapping BACs hybridized to both sides of the breakpoint and reduced the breakpoint critical region to less than 200Kb.

POSTER 72 - SPERM ANEUPLOIDY IN PL/J MICE: A COMPLEX PHENOTYPE

Prof. Mary Ann Handel
University of Tennessee
Dept. Biochem.and Cellular & Molecular Biology
University of Tennesee
Knoxville, TN
37996-0840
USA

Co-Authors: Pyle, A.

Institutions: Dept. Biochem.and Cellular & Molecular Biology, University of Tennesee, Knoxville, TN 37996-0840

We previously showed that sperm from PL/J males have increased frequencies of both sperm head abnormalities and sperm aneuploidy compared to C57BL/6J sperm.  This trait is complex phenotypically.  During prophase, spermatocytes of PL/J mice exhibit premature chromosome desynapsis.  Abnormalities are more apparent during the first meiotic division, where 34.7% of the PL/J spermatocytes exhibit aberrant spindle morphology (compared to 5.7% for C57BL/6J spermatocytes).  Spindle abnormalities include monopolar spindles, split spindle poles and incomplete spindle formation.  Staining with antibody to pericentrin reveals abnormal numbers of centrosomes in PL/J spermatocytes.  Thus increased frequency of sperm aneuploidy in PL/J males is associated with, and may be caused by, aberrant centrosome and spindle formation.  The traits of sperm aneuploidy and aberrant head morphology are also complex genetically.  F1 progeny of a cross between PL/J and C57BL/6J do not exhibit a higher frequency of either sperm aneuploidy or sperm-head morphology aberrations, as would be expected for a dominant trait.  Among progeny of a backcross of the F1 to PL/J, none of 16 males assessed exhibited elevated frequencies of either sperm aneuploidy or sperm-head morphology abnormalities.  Thus the traits are apparently inherited in a complex manner, with several genes and/or modifiers affecting the generation of sperm aneuploidy and abnormal sperm-head morphology.  (Supported by NIH HD33816 to MAH)

POSTER 73 - FUNCTIONAL AND GENOMIC ANALYSIS OF THE SIX GENE FAMILY

Dr Isabel Hanson
Dept. of Medical Sciences
University of Edinburgh
Molecular Medicine Centre
Western General Hospital
Crewe Road
Edinburgh
EH4 2XU
UK

Co-Authors: Brown AG

Institutions: Medical Genetics Section, University of Edinburgh, Molecular Medicine Centre, Western General Hospital,

Members of the SIX gene family are involved in a variety of developmental processes in vertebrates and invertebrates. All SIX genes encode two highly conserved motifs, a homeodomain that mediates DNA binding, and a SIX domain that mediates protein-protein interactions. With the near completion of various genome projects, it is now clear that Drosophila has three SIX genes (sine oculis, optix and D-Six4) while mammals have six (SIX1-6). Phylogenetic analysis of Drosophila and mammalian amino acid sequences shows that the proteins fall into three clear subgroups. An intriguing feature of the mammalian SIX genes is their genomic organisation; five of the six genes are clustered. We review the detailed organisation of the human genes, as recently revealed by the Human Genome Sequencing Project. Despite extensive amino acid homology within the SIX-domain, SIX proteins differ dramatically in their interactions with transcriptional co-factors encoded by the EYA gene family. We report work in progress which shows that while SIX1 interacts strongly with EYA2 and EYA4 in a yeast two-hybrid assay, SIX3 interacts with neither. This implies that there are clear functional differences between the subgroups of SIX proteins.

POSTER 74 - THE BPA MUTATION IS ASSOCIATED WITH PLACENTAL DEFECTS

Dr. Gail Herman
Children's Research Institute
700 Children's Dr.  Rm W403
Columbus,OH
43205
USA

Co-Authors:  Wang J, Peters J, Cattanach B, Kelley RI

Institutions: Children's Research Institute, Mammalian Genetics Unit and Mouse Genome Centre,  Kennedy Krieger Institute

The X-linked dominant male-lethal mouse mutation bare patches (Bpa) results from mutations in Nsdhl. The Nsdhl enzyme is involved in the removal of C-4 methyl groups in the cholesterol biosynthetic pathway. Male embryos for several Bpa alleles die during midgestation (day 10.5-13.5 pc). Since cholesterol levels in affected male embryos at the time of death are normal, the pathogenesis of the male lethality must be caused by another mechanism. Placentas from affected male embryos are smaller than those of normal littermates (p < 0.001), and the labyrinthine layer of the placenta appears thinner, disorganized, and has fewer fetal vessels by PECAM staining. There are also statistically significant differences in placental thickness between affected male and affected female placentas. Since most cells in the female rodent placenta undergo preferential inactivation of the paternal X Chr, we believe that cells derived from allantoic mesoderm that undergo random X-inactivation are responsible for or contribute to the male lethality. Finally, we have been unsuccessful in attempts to generate transgenic mice that overexpress Nsdhl. Preliminary data from experiments in which a tyrosinase minigene was coinjected with an Nsdhl cDNA suggest that such overexpression may be lethal during embryogenesis. A possible explanation could include feedback regulation of isoprenoid synthesis earlier in the cholesterol biosynthetic pathway.

POSTER 75 - A NEW MATERNALLY EXPRESSED TRANSCRIPT WITHIN THE Gnas IMPRINTING CLUSTER

Rebecca Holmes
MRC Mammalian Genetics Unit
Harwell
Didcot
OX11 0RD
UK

Co-Authors: Williamson CM, Skinner JA, Beechey CV, Peters J.

Institutions: MRC Mammalian Genetics Unit

The Gnas cluster on Chromosome (Chr) 2 of the mouse produces a number of imprinted and alternatively spliced transcripts.  On the sense strand these include Nesp, which is maternally expressed, Gnasxl and Gnas exon1a, which are paternally expressed.  All three splice into exon 2 of Gnas itself and act as alternative first exons for Gnas.  Gnas itself is biallelically expressed in most tissues and encodes a G protein alpha sub-unit; Gnasxl encodes an extra large form of the G protein and Nesp a neuroendocrine secretory protein whereas the Gnas exon 1a transcript appears to be non-coding.  Genomically Gnas lies at one end of the cluster with Gnas exon 1a, Gnasxl and Nesp 2kb, 30 kb and 45kb upstream, respectively.  Analysis of the 15kb genomic sequence between Nesp and Gnasxl has revealed a number of ESTs.  These are organised into four groups that lie 2, 4, 6.5 and 10kb downstream from Nesp.  Their imprinting status has been analysed in mice carrying either two maternal copies and no paternal copies (MatDp) or two paternal copies and no maternal copies (PatDp) of distal Chr 2.  A transcript was found in the MatDp(dist2) mice which was not present in the PatDp(dist2) mice.  Preliminary analysis suggests this new maternal transcript links the groups of ESTs lying between Nesp and Gnasxl.  One of the ESTS links to exon 2 of Gnas and thus would appear to represent a new alternative first exon of Gnas.

POSTER 76 - CHARACTERISATION OF EHOX, A NOVEL HOMEOBOX-CONTAINING GENE

Dr Melany Jackson
University of Edinburgh
John Hughes Bennett Laboratory
Western General Hospital
Crewe Road
Edinburgh
EH4 2XU
Scotland

Co-Authors: 1) Baird J, 2) Cambray N,  2) Ansell J, 1)Graham G,  2) Forrester L.

Institutions: 1) The Beatson Institute, 2) John Hughes Bennett Laboratory, University of Edinburgh

The differentiation of ES cells as embryoid bodies in vitro is a valuable model for embryonic development and for the functional characterisation of genes involved in this process. To identify novel genes that may be involved in embryogenesis we generated a subtracted cDNA library from RNA isolated from day 5 and day 3 embryoid bodies. One of these cDNAs, (ehox) encodes a novel gene that contains a homeodomain and was  identified as a potentially interesting developmentally-regulated gene.   We used an episomal overexpression system as an initial screen to assess the function of ehox in ES cells. Plasmids carrying the ehox cDNA in sense or anti-sense orientations were transfected into ES cells and colonies were selected in puromycin.  Transfection experiments indicated that  high levels of eHOX  expression was incompatible with an undifferentiated ES cell phenotype.  In addition, we observed that ES cells expressing anti-sense ehox maintained a stem cell phenotype  in low concentrations of LIF whereas ES cells expressing sense ehox, EGFP or the empty vector, differentiated in a comparable manner to parental ES cell lines. ES cells expressing anti-sense ehox are also unable to differentiate into beating cardiomyocytes in contrast to eHOX or GFP-expressing cells.  Our data therefore suggest that eHOX is required either for the initial differentiation of pluripotent ES cells or for the survival of differentiated cell types.

POSTER 77 - USING TRANSPOSON INSERTIONS IN LARGE DNA CLONES TO ANALYZE GENE EXPRESSION

Dr. Timothy Jinks
MRC-NIMR
Division of Dev. Neurobiology
The Ridgeway
Mill Hill
London NW7 1AA
UK

Co-Authors: 1) Martinez MT, 2) Sedgwick S, 3) Krumlauf R.

Institutions: 1)MRC-NIMR, Division of Dev. Neurobiology, 2)MRC-NIMR, Division of Yeast Genetics, 3) Stowers Institute for Medical Research

Large insert clones of genes and gene complexes are increasingly being used to analyze gene regulation and gene function.  These large insert clones are often difficult to manipulate due to the size and complexity of the sequences in the clone.  New molecular biology techniques are needed to modify these clones for use in experimentation.  We have taken advantage of transposon-mediated integration to create a series of individual random inserts of a reporter gene in Hoxb BACs.  The series of modified BACs were analyzed by mouse transgenesis to reveal the reporter¹s expression pattern.  We have analyzed the reporter¹s expression for each insertion site and have correlated expression with the precise location in the Hoxb complex.  This has allowed us to establish the range over which enhancers influence gene expression across the complex and also the limit of action of the enhancers beyond the 3¹ end of the cluster.  This technique is useful for the systematic analysis of large clones to scan for gene regulatory activity.  In addition we have engineered a new transposon-based reporter that provides further utility for analysing large clones by introducing loxP and FRT recombination sites.  These sites permit precise rearrangement of the BACs to be made as well as targeted integration of other DNA sequences.  These new tools are proving useful for our analysis of Hoxb gene regulation and should prove useful more generally for the analysis of large clones.

POSTER 78 - FAILURE OF URETERIC BUD INVASION: A NEW MODEL OF RENAL AGENESIS IN MICE

Dr Tomomi Kamba
Kyoto University
Yoshida-Konoe-cho, Sakyo-ku
Kyoto
606-8501
Japan

Co-Authors: 1)Higashi S, 1)Kamoto T, 2)Shisa H, 3)Yamada Y, 1)Ogawa O, 3)Hiai H

Institutions: 1)Department of Urology, Kyoto University Graduate School of Medicine 2) Saitma Cancer Institute, 3)Department of Pathology and Biology of Pathology and Biology of Diseases, Kyoto University Graduate School of Medicine

FUBI (Failure of Ureteric Bud Invasion) is a highly inbred strain of mouse with a high spontaneous incidence of unilateral or bilateral renal agenesis(60%). Bilateral renal agenesis is lethal within 2 days after birth. The primary defect of FUBI is failure of the ureteric bud to penetrate into the metanephric mesenchyme at around embryonic day 11, resulting in apotosis of metanephric cells and leading to renal agenesis on the affected side. The metanephros seemed to be normal because coculturing of the FUBI metanephros with homologous spinal cord induced differentiation of the rudiment, but coculturing with the homologous ureteric bud frequently did not. Genetic analysis revealed that more than two genes were involved in this malformation and we mapped one of the modifier loci, fubi1, on chromosome 2, at about 65cM from the centromere. In this region, there are two possible candidate genes, Wilms tumour 1 and formin, that play important roles in kidney development. Some of formin mutants shared a similar phenotype with FUBI; however, there was no difference in the expression of formin in embryonic kidneys between FUBI and control NFS/N mice. Studies of fubi1 congenic mice indicated that interaction of two or more loci is essential for the FUBI phenotype.

POSTER 79 - TRANSGENIC MICE EXPRESSING HUMAN ZAC, A CANDIDATE GENE FOR THE IMPRINTED DISORDER TNDM

Dr. Gavin Kelsey
MRC Senior Fellow
The Babraham Institute
Developmental Genetics Programme
Cambridge
CB2 4AT
United Kingdom

Co-Authors: Ma D, Smith RJ, Arnaud P, Konfortova G, Dean WL

Institutions: Developmental Genetics Programme, The Babraham Institute

As the result of a methylation-based screen for imprinted genes in the mouse, we identified Zac1 as an imprinted gene with paternal expression. The Zac1 CpG island has maternal-allele methylation and may be involved in controlling imprinting of the locus, as methylation is present in oocytes but not sperm. Zac1 encodes a zinc finger protein which promotes apoptosis and cell cycle arrest. The gene resides on Chr. 10, a chromosome without recognised imprinting effects. The human homologue (6q24-q25) is a candidate for the imprinted disorder transient neonatal diabetes mellitus (TNDM). TNDM is a developmental disease of insulin production, characterized by intrauterine growth retardation, insulin dependence in infants and a 60% risk of developing permanent diabetes. TNDM occurs with paternal duplication of 6q, implicating overexpression of ZAC in the disease mechanism. Uniparental disomy for mouse Chr. 10 is not associated with a phenotype similar to TNDM. To account for this phenotypic discrepancy and investigate a potential animal model for TNDM, transgenic mice with PACs containing human ZAC have been made. In all transgenic lines examined, human ZAC is imprinted. RT-PCR and in situ analyses show that offspring from transgenic males express human ZAC widely, including the pancreas, pituitary and adrenal gland. Further work will examine blood glucose homeostasis, pancreatic beta cell function and apoptosis in this transgenic model.

POSTER 80 - CHARACTERIZATION OF IMMUNOGLOBULIN HEAVY SCKIN REPERTOIRE AND RNA EXPRESSION PROFILE OF SPLENIC AND PERITONEAL B-1a LYMPHOCYTES IN l2315 TRANSGENIC MICE

 

Karsten Kretschmer
Molecular Immunology, GBF,
German Research Centre for Biotechnology
Mascheroder Weg 1
D-38124
Braunschweig

Germany

Co-Authors: Reinhard Hoffmann

Institution:   Molecular Immunology, GBF German Research Centre for Biotechnology, 2) Max-von-Pettenkofer-Institut, Dept of Bacteriology

Recently a new mouse line – L2 – was established which is transgenic for the l2 immunoglobulin light chain of the plasmacytoma MOPC315.  These mice are characterized by a high and age-independent expression of the transgene, which results in nearly complete abrogation of rearrangment  of endogenous light chain isotypes.  One of the most interesting features of L2 mice is the predominance of a CD5+ B-1 cells in both peritoneal cavity and spleen.  Therefore L2 mice represent a simplified experimental model to study the characteristics and the antibody repertoire of these B-cell subpopulations.  Using transgenic L2 and control mice, we performed a comparative sequence analysis of the DJ rearrangements of splenic and peritoneal B-1a lymphocytes from adult L2 mice and B cells derived from fetal/neonatal liver or in vitro PreB cell cultures.  Our data suggest that strong selective forces act at each particular location shaping the repertoire in an organ specific way.

In order to elucidate the molecular feature that in addition to the above elective forces act on these mature B-1a cell populations we performed mouse gene expression microarray analysis and compared splenic and peritoneal cavity derived B-1a cells with other B cell subpopulations.  These data will help to understand the molecular events that these distinct B-1a cell subsets have to undergo in order to function as an efficient defense bastion against pathogens.

POSTER 81 - FRINGE GENE EXPRESSION IN THE DEVELOPING MOUSE LIVER

Dr. Kathleen Loomes
The Children's Hospital of Philadelphia
34th St. and Civic Center Blvd.
Philadelphia, PA
19104
USA

Co-Authors: 2) Taichman DB, 1) Glover CL, 3) Baldwin HS, 4) Oakey RJ

Institutions: 2)Division of Pulmonary Medicine, University of Pennsylvania School of Medicine. 1)Division of Gastroenterology and Nutrition, The Children's Hospital of Philadelphia. 3)Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine. 4)Division of Human Genetics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine

The discovery that Jag1 is the disease gene for Alagille syndrome (AGS), a developmental disorder with bile duct paucity as a major feature, implicates Jag1 and the Notch signaling pathway in bile duct development. The Fringe genes, Manic fringe (Mfng), Lunatic fringe (Lfng) and Radical fringe (Rfng), directly modify Notch and affect its ability to respond to signaling by a ligand. We hypothesize that the Fringe genes, as modifiers of the Notch pathway, play an important role in liver and bile duct development.  RNA and cDNA were prepared from staged mouse livers ranging from 11.5 dpc to 12 weeks postnatal. Gene expression was analyzed using real time quantitative RT-PCR. All reactions were performed in multiplex with GAPDH. Lfng expression is low relative to GAPDH at 11.5 dpc, gradually increasing to a peak of 5 times the initial value at 13.5 dpc, and then falling by 18.5 dpc. Similarly, Rfng expression peaks at 13.5 dpc at about 2.5 times the initial value. In contrast, Mfng expression remains relatively constant throughout development and then peaks postnatally at 12 weeks at 5 times the initial value. Each fringe gene demonstrates a unique pattern of expression in the developing liver when measured by real time quantitative RT-PCR. Lfng and Rfng expression both peak at 13.5 dpc, just prior to the onset of bile duct development.  Studies are ongoing to define fringe expression spatially in the liver and to analyze the fringe knockout mice for any liver abnormalities.

POSTER 82 - USING SEGMENTALLY TRISOMIC MICE TO DETERMINE THE MECHANISM OF GENE ACTION RESPONSIBLE FOR DOWN SYNDROME CEREBELLAR PHENOTYPES

Ms. Lisa Olson
Johns Hopkins University School of Medicine
725 N. Wolfe St.
Biophysics 203
Baltimore, MD 21205
USA
410-955-6624 (phone)
410-614-8731 (fax)

Co-Authors: 1) Olson LE, 1) Baxter LL, 2) Carlson EJ, 2) Epstein CJ, 1) Reeves RH

Institutions: 1) Johns Hopkins University School of Medicine, Baltimore, MD  2) University of California at San Francisco. 

In rare individuals with chromosomal translocations, Down syndrome (DS) is caused by partial trisomy of chromosome (Chr) 21.  Correlation of the smallest regions of overlap in such individuals also displaying the same characteristics of DS has led to the hypothesis that many aspects of the phenotype arise from dosage imbalance of one or a few genes.  An alternative hypothesis asserts that DS phenotypes result from small effects of hundreds of genes at dosage imbalance.  Segmentally trisomic mice, which produce dosage imbalance for different numbers of Chr 21 gene orthologs, provide sensitive models in which to test this hypothesis.

By comparing mouse Chr 16 and human Chr 21, we know that Ts1Cje and Ts65Dn mice are respectively trisomic for 85 and 124 of the 225 catalogued Chr 21 genes.  Ts65Dn mice, like individuals with DS, have reduced volume and cellular density in the cerebellum (Baxter et al., 2000); thus, trisomy for roughly half of the genes on Chr 21 is sufficient to produce this DS phenotype.  The cerebellum of the Ts1Cje mouse is affected more mildly than that of the Ts65Dn mouse.  Although cerebellar volume is reduced to the same extent as Ts65Dn mice (88% of euploid), granule cell density is substantially less affected in Ts1Cje (90% of euploid) as compared to Ts65Dn (76%).  The Ts1Cje mice did not show the reduced Purkinje cell density found in Ts65Dn.  This graded severity of the cerebellar phenotype with decreasing amounts of trisomy supports the hypothesis that multiple genes contribute to this phenotype of DS.

Baxter, L. L., T. H. Moran, J. T. Richtsmeier, J. Troncoso, and R. H. Reeves.  2000.  Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse.  Hum Mol Genet 9: 195-202.

POSTER 83 - AN ABD-B CLASS HOX/PBX RECOGNITION SEQUENCE IS EQUIRED FOR EXPRESSION FROM THE MOUSE REN-1C GENE

Li Pan
Dept of Molecular & Cellular Biology
Roswell Park Cancer Institute
Buffalo
New York
14263

Co-Authors: Xie Y, Black TA, Jones CA, Pruitt SC, Gross KW

Institutions: Dept of Molecular & Cellular Biology, Roswell Park Cancer Institute

Expression from the mouse Ren-1c gene in As4.1 cells is dependent on a proximal promoter element (PPE), located at approximately -60, and a 241 base pair (bp) enhancer region, located at -2625, relative to the transcription site.  The PPE (TAATAAATCAA) is identical to a consensus Hox/Pbx binding sequence. Further, Pbx1b was shown to be a component of a PPE specific binding complex present in nuclear extracts from As4.1 cells. The binding affinities of different paralog Hox members to the PPE were examined in the absence or presence of Pbx1b. HoxB6, B7 and C8 failed to bind the PPE alone but showed weak affinity in the presence of Pbx1b. In contrast, HoxD10, and to a lesser degree Hox B9, bound the PPE with high affinities regardless of whether Pbx1b was present. Abd-B Hox members, including HoxD10, A10, A9, B9 and C9, are expressed in As4.1 cells. The ability of Hox and Pbx1b to from a ternary complex with Prep1, on the PPE, is also demonstrated both in vivo and in vitro. Point mutations in either the Hox or Pbx half site of the PPE disrupted the formation of the Hox/Pbx complex and dramatically decreased transcriptional activity of the Ren-1c gene, demonstrating that both the Hox and Pbx half sites are critical for mouse renin gene expression. These results strongly implicate Abd-B class Hox genes, and their co-factors, as major determinants of the sites of renin expression.

POSTER 84 - WHAT IS THE ROLE OF NESPAS, A GENE THAT RUNS ANTISENSE TO NESP AND GENERATES A MULTITUDE OF IMPRINTED TRANSCRIPTS?

J Peters
MRC Mammalian Genetics Unit
Harwell
Didcot
Oxon
OX11 0RD

Co-Authors: 3)Peters J, 3)Ball ST, 3)Skinner JA, 3)Nottingham W, 1)Plagge A, 1)Kelsey G, 2)Hayes C, Hacker T and Williamson CM

Institutions: 3)MRC Mammalian Genetics Unit, 1)The Babraham Institute, 2) Current address Merck, Sharp and Dohme,

Within the imprinted Gnas complex in distal mouse chromosome 2 there is a gene called Nesp transcribed in the sense direction.  Nesp has maternal specific expression. Several antisense RNAs, all of which are imprinted, paternally expressed, and non-coding, overlap the Nesp exons. These antisense RNAs are called Nespas. In addition to an unspliced form, there are five alternatively spliced forms that are up to 1.4kb in length. The splice variants, and probably the unspliced form as well, start approximately 13 kb downstream of Nesp in a region of maternal methylation .

An increasing number of imprinted genes have antisense transcripts associated with them. One of the issues in imprinting is to define their role. One thought is that they may function as cis-acting regulators of the sense gene they overlap, and another is that they are spurious transcripts with no role of their own.  In newborn heart Nesp is maternally expressed and Nespas is paternally expressed, which would be consistent with the idea that the antisense controls expression of the sense transcript. However, although Nesp and Nespas are also expressed from opposite parental alleles in mid gestation embryos, Nesp expression is primarily seen in the somites and developing vasculature whereas Nespas is mainly detected in the developing limb, particularly in the progress zone. These observations suggest that Nespas may not be involved in regulating the imprinting of Nesp and that it has an alternative role. In order to explore the role of Nespas further, a gene knock-out experiment is underway.

POSTER 85 - MOLECULAR CHARACTERIZATION OF A MOUSE MODEL FOR POLYCYSTIC KIDNEY DISEASE

Sarah J. Price
Department of Microbiology, Immunology, and Molecular Genetics
Joan C. Edwards School of Medicine
Marshall University
Huntington
WV

Co-Authors: Elizabeth C. Bryda

Institutions: Department of Microbiology, Immunology, and Molecular Genetics.  Joan C. Edwards School of Medicine, Marshall University

The mouse juvenile congenital polycystic kidney disease (jcpk) gene causes an aggressive, early-onset form of polycystic kidney disease (PKD) characterized by fluid-filled cysts in all segments of the nephron.  When inherited in an autosomal recessive manner, this disease is the most severe of all the known mouse mutations for PKD, with death occurring within 10 days after birth. Expression studies have shown that the jcpk gene is expressed in a variety of tissues including kidney, heart, lung, ovary, testis and brain.  This gene is also expressed in the developing embryo as early as 7 days post coitum.

The pathogenesis of cyst formation in jcpk homozygous mice is unknown. To unravel some of the molecular mechanisms underlying cystogenesis in this model, cDNA microarray analysis was performed to simultaneously profile expression differences between kidney mRNA from a homozygous jcpk animal versus that of a wildtype littermate.  Over 1500 different genes were profiled and a subset of both up- and down-regulated genes were chosen for further analysis. Currently, efforts are focused on using real-time PCR to determine whether the differential expression of these genes are primary factors involved in cystogenesis or are secondary to the pathology of cyst formation.

POSTER 86 - DELTA/NOTCH SIGNALLING IS REQUIRED FOR PROPER LEFT-RIGHT ASYMMETRY IN MICE

Dr. Gerhard Przemeck
GSF-National Research Centre
Institute of Experimental Genetics
Ingolstaedter Landstr. 1
Neuherberg
D-85764
Germany

Co-Authors: 1) Heinzmann U, 2) Beckers J, 2)Hrabé de Angelis M

Institutions: 1) GSF - Institute of Pathology; 2) GSF - Institute of Experimental Genetics

Axes formation is a fundamental process of early embryonic development. In addition to the antero-posterior and dorso-ventral axes, the determination of the left-right (L/R) axis is crucial for the proper morphogenesis of internal organs and is a distinctive feature of vertebrates. Genes known to be required for the normal establishment and/or maintenance of L/R asymmetry in mammals include, for example, components of the TGF-b family of intercellular signalling molecules and genes required for node cilia function. Delta/Notch signalling, which had not been implicated in this morphogenetic process so far, is known to be required, for example, for proper somitogenesis, normal differentiation of pancreatic endocrine cells and inner ear sensory development. Here, we report that Delta/Notch signalling is also required for normal L/R determination in mice. We show that the loss-of-function of the Delta1 gene causes a situs ambiguous phenotype including randomisation of the orientation of heart looping and embryonic turning. A possible cause for this L/R defect in Delta1 mutant embryos is a failure in the development of proper midline structures. These originate from the node. Interestingly, we find that Delta/Notch signalling is also required for the proper differentiation of node cells and node morphology.

POSTER 87 - The mutations Diminuendo and Catweasel result in changes in hair cells in the sensory patches of the mouse inner ear

Elizabeth Quint
MRC Institute of Hearing Research
University Park
Nottingham
NG7 2RD

Co-Authors: 1)Quint E, 2)Fuchs H, 3)Balling R, 2) de Angelis H, 1) Steel KP

Institutions: 1)MRC Institute of Hearing Research, 2)Institute of Experimental Genetics, GSF National Research Center for Environment and Health, 3)Institute for Mammalian Genetics, GSF National Research Centre for Environment and Health

Although many human deafness loci are known, few of these currently have a homologous animal model.  Recent mouse mutagenesis initiatives have however provided new mutations that promise to be useful models of human deafness and vestibular dysfunction.  We have begun detailed phenotypic and genotypic analyses on two of these new mutant lines, Diminuendo and Catweasel with initial phenotypic assessment focussed on the integrity and patterning of the inner ear sensory patches using scanning electron microscopy.

Mutant mice were obtained from N-ethyl-N-nitrosourea (ENU) mutagenesis, and F1 offspring of mutagenised males were screened for new dominant mutations that affect hearing and/or balance.  The mutant diminuendo exhibits a progressive hearing loss whereby the Preyer reflex (an ear flick in response to sound), which is present from about 2 weeks of age, is lost postnatally.  This corresponds to abnormal organisation of the hair-cell bundles in the early postnatal diminuendo organ of Corti (observed at P3) and a progressive basal to apical loss of inner and outer hair `cells (over the following 4-6 weeks).  The mutant catweasel exhibits behavioural abnormalities, including headtossing, which is suggestive of a vestibular deficit.  In mutant mice there appears to be a regional reduction in hair cell number in the utricle and the stereocilia of existing hair cells appear to be less numerous and thicker than in the littermate controls. 

Backcrosses have been set up to map diminuendo and catweasel onto the mouse genome and, combined with further analysis of the hair cell changes described above, should help establish appropriate candidate genes for these mutations.

This work is supported by the MRC, Defeating Deafness and the EC (contact number QLG2-CT-1999-00988)

POSTER 88 - THE HOMEOBOX GENE EMX2 IS RESPONSIBLE FOR HEARING DEFECTS IN THE DEAF MOUSE MUTANT PARDON

Charlotte Rhodes
MRC Institute of Hearing Research
University Park
Nottingham
NG7 2RD

Co-Authors: 1)Rhodes CR, 2)Parkinson N, 2)Tsai H, 3)Brook D, 3)Robinson T, 2)Brown SDM,

1)Steel KP

Institutions: 1)MRC Institute of Hearing Research, 2)MRC Mammalian Genetics Unit and UK Mouse Genome Centre, 3)Institute of Genetics, Queens Medical Centre

A phenotypic approach has been adopted in the mouse to identify molecules involved in ear development and function.  Mutant mice were obtained using N-ethyl-N-nitrosourea mutagenesis and were screened for new dominant mutations that affect hearing and/or balance.  The genetic and phenotypic analysis of one of these mutants, Pardon, is described.

Pardon mice (Pdo/+) are identified by their lack of a Preyer reflex (ear flick) following the delivery of a 20kHz 90dB SPL tone burst.  Dissection of the middle ear has revealed morphological defects of the ossicles which disrupt the ossicular chain resulting in a conductive hearing loss.  Physiological recordings of cochlear responses of Pardon mutants show very raised thresholds which could not be accounted for by a conductive hearing loss alone.  Analysis of the surface of the organ of Corti by scanning electron microscopy has revealed an increase in numbers of outer hair cells.  Pdo/+ mutants appear to be healthy and fertile but Pdo/Pdo mutants die shortly after birth.   The reason for their demise requires further investigation.

Pdo was mapped to mouse chromosome 19 between markers D19Mit137 and D19Mit6.  The homeobox gene, Emx2 was located in this region and was considered a strong candidate for Pdo due to its expression in the branchial arches and the otic vesicle. Sequence analysis of the Emx2 gene in Pdo mutants revealed a missense mutation causing a non-conservative amino acid change in the homeodomain. This work is supported by the MRC, Defeating Deafness and a grant from the EC (contract number QLG2-CT-1999-00988).

POSTER 89 - IMPORTANCE OF EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN ESTABLISHMENT OF ADENOMAS AND MAINTENANCE OF CARCINOMAS DURING INTESTINAL TUMORIGENESIS

Reade Bruce Roberts
University of North Carolina Genetics Department
Threadgill Lab
UNC Dept of Genetics
11-109 Lineberger Cancer Center
Chapel Hill, NC
27599-7264
USA

Co-Authors: Min L.; Washington MK, Olsen, SJ, Coffey RJ, Threadgill DW

Institutions: Departments of Cell Biology, Pathology, Medicine, and Vanderbilt-Ingram Cancer Center, Vanderbilt University, and Veterans Affairs Medical Center

We utilized the hypomorphic Egfrwa2 allele to genetically examine the impact of impaired Egfr signaling on the ApcMin mouse model of Familial Adenomatous Polyposis (FAP). Transfer of the ApcMin allele onto a homozygous Egfrwa2 background results in up to a 97-percent reduction in intestinal polyp number relative to ApcMin mice carrying a wild-type Egfr allele. This Egfr effect may be synergistic with the actions of the Modifier-of-Min (Mom1) locus. Surprisingly, the size, expansion, and pathological progression of the polyps appear Egfr-independent. Histological examination of the ilea of younger animals revealed no differences in the numbers of nuclear b-catenin positive cells, a marker for Apc loss, or of microadenomas, the presumptive precursor lesions to gross intestinal polyps. Pharmacological inhibition with EKI-785, an Egfr tyrosine kinase inhibitor, produced similar results in the ApcMin model. These data suggest that normal Egfr activity is required for establishment of intestinal tumors in the ApcMin model between initiation and subsequent expansion of initiated tumors. The role of Egfr signaling during later stages of tumorigenesis was examined using nude mice xenografts of two human colorectal cancer (CRC) cell lines. Treatment with EKI-785 produced a dose-dependent reduction in tumor growth, suggesting that Egfr inhibitors may be useful for advanced CRC treatment.

POSTER 90 - GENOME  ANALYSIS OF CRITICAL REGION OF A MOUSE PREAXIAL POLYDACTYLY MUTATION, HEMIMELIC EXTRA TOES (HX) (Hx)

Dr Tomoko Sagai
Mammal. Genet. Natl. Inst. Genet.
1111 Yata
Mishima
411-8540
Japan

Co-Authors: 1) Masuya H, 2) Shimizu K, 3) Yada Y, 4) Tamura M, 5) Shiroishi T

Institutions: 1)Riken Gsc, 2)Nihon University, 3)Ochanomizu University, 4)Kyoto University, 5) Mammalian Genetics Natl.Inst.Genet.

Preaxial polydactyly of mouse hemimelia luxate member is associated with an ectopic expression of Shh in the anterior limb mesenchyme. Little is known for the molecular mechanism of such a misregulation of shh. One of the member, hemimelic extra-toes (Hx) is a dominant mutation characterized by preaxial polydactyly and shortening of the radius, tibia and talus. Hx has been mapped to the extreme vicinity of shh. Recently, Lmbr1 encoding a novel transmembrane receptor, has been served as a possible candidate of Hx. Lmbr1 is a mouse homolog of C7orf2, which was identified as a candidate of the limb deformity mapped in the human syntenic region, 7q36. Although Lmbr1/C7orf2 has been reported to have a role in the limb development, no mutation that causes preaxial polydactyly has been identified both in human and mouse. In this study, we constructed precise genetic and physical maps and localized Lmbr1 in the critical region. This study, however, did not reveal any mutation in the Lmbr1coding region of Hx mutant. Furthermore, we did not detect large alteration of the Lmbr1 expression level in the developing limb buds of Hx homozygote. Therefore, it is still circumstantial that Lmbr1 is the direct causative gene for Hx.

POSTER 91 - REQUIREMENT FOR THE HOX-COFACTOR PBX1 IN SKELETAL PATTERNING AND NORMAL ORGANOGENESIS

Professor Licia Selleri
Institute of Genetic Medicine
"Program of Genetics and Cell Biology of Cornell/Sloan Ketttering"
Cornell University Medical School
Whitney W-406, 1300 York Avenue
New York
NY 10021
USA

Co-Authors: 1) Depew M, 1)Rubenstein J, 2) Kim S, 3) DiMartino J, 3) Cleary M

Institutions: 1)Nina Ireland Laboratory, UCSF, 2)Department of Developmental Biology, Stanford University, 3)Department of Pathology, Stanford University

Pbx1, homologue of Drosophila EXD, is a TALE (three amino acid loop extension) homeodomain protein.  As a heterodimer with other TALE proteins, Pbx1 binds DNA with Hox proteins that contain tryptophan-bearing dimerization motifs. To present, the lack of Pbx1 germline mutants has compromised the analysis of its in vivo contributions to mammalian development.  We generated and characterized Pbx1-deficient mice.  Pbx1 is an essential gene, whose loss results in embryonic lethality with hypoplasia or aplasia of multiple organs (Selleri et al., Development 126, in press).  A role in patterning is evidenced by widespread defects of the skeleton, including transformation of skeletal elements of the second branchial arch. Absence of Pbx1 results in premature chondrocyte differentiation and reduced proliferation. Marked differences in the proliferative state of chondrocytes were observed by Bromodeoxyuridine (BrdU) in vivo labeling of wt and Pbx1-/- embryos. These studies reveal a novel function of Pbx1 in temporally coordinating the extent of chondrocyte proliferation with terminal differentiation. They parallel our observations that the hematopoietic defect in Pbx1-/- embryos results from deficiencies in the proliferative expansion of hematopoietic progenitor cells (DiMartino, Selleri et al., Blood 98, in press).  Interestingly, the pancreatic hypoplasia and defects in exocrine and endocrine cell differentiation in Pbx1-/- embryos (Kim, Selleri et al., submitted) are also characterized by a reduction of pancreatic cell proliferation. Thus, Pbx1 may serve analogous roles to orchestrate the growth properties of progenitors at specific developmental stages, within multiple cellular lineages. 

POSTER 92 - BEHAVIORAL ANALYSIS OF MICE THAT ARE DEFICIENT IN AP-3 OR IN MURINE HPS1

Mrs Eunju Seong
University of Michigan
Mental Health Research Institute
University of Michigan
205 Zina Pitcher Place
Ann Arbor, MI
48109-0720
USA

Co-Authors: Burmeister M

Institutions: Mental Health Research Institute, University of Michigan

Hermansky Pudlak syndrome (HPS), a disorder characterized by hypopigmentation and delayed blood clotting, is explained by the inability of melanosomes, lysosomes, and storage granules of platelets to store their contents. Several years ago, in both HPS patients and the mouse mutant pale ear (ep), responsible mutations were found in a novel gene of unknown function, called HPS1. Recently, mutations in HPS patients and another mouse mutant were also identified in a gene encoding a subunit of the adaptor-like complex AP-3. The fact that AP-3 is involved in vesicle trafficking from the trans-Golgi network to endosomal compartments and a subset of synaptic vesicles explains well the cellular defects of HPS patients. Based on these findings, we hypothesized that the HPS protein is involved in the biological pathways of AP-3 and tested this hypothesis by constructing a mouse double mutant for HPS1 and AP-3. In the double mutant line, the introduction of ep mutation, which by itself only affects tail and ear pigmentation, significantly diluted the coat color of the AP-3 mouse mutant, supporting the given hypothesis. On the other hand, neither the HPS patients nor the ep mice have ever been reported to have neurological or behavioral abnormalities. However, here we report that in the open field test the double mutant mice displayed a synergistic increase in turning bias compared to each single mutant line, suggesting a neurological role of the HPS1 protein.

POSTER 93 - VEZATIN, A NOVEL TRANSMEMBRANE PROTEIN, THAT BRIDGES MYOSIN VIIA TO THE CADHERIN-CATENINS COMPLEX : EXPRESSION PATTERN DURING EARLY MOUSE DEVELOPMENT

Dr Marie-Christine Simmler
CNRS/UMR955 INRA
Ecole vétérinaire d'Alfort
7 avenue du général de Gaulle
Maisons-Alfort
94704
France

Co-Authors: 1)Daric V, 2)El-Amraoui A, 1)Guillaud L, 1)Panthier JJ, 2)Petit C, 1)Simmler MC

Institutions: 1) UMR955 INRA, Ecole vétérinaire d'Alfort.  2) URA1968 CNRS, Institut Pasteur

Early embryonic stages in the mouse are potential models for studying the role of cell adhesion molecules in polarised epithelium formation, during development. The compaction of the embryo is known to be mediated by cell adhesion molecules such as cadherins and catenins. It involves the actin cytoskeleton and requires the proper assembly of cell junctions. Cell adhesion molecules are also essential for the trophectoderm epithelium formation which represents the first visible differentiation process during mouse pre-implantation embryonic development. In vitro, embryonic stem (ES) cells are cells that may be differentiated to organized structures known as embryoid bodies (EBs) containing an outer endodermal layer and an inner ectodermal layer, separated by a basal lamina. These cells are polarized with microvilli on the apical membrane, a basement membrane underlying  the epithelial cell layer. Vezatin, recently identified as a ligand to myosin VIIA, is an ubiquitous component of adherens cell-cell junctions, interacting with the cadherin-catenins complex (Kussel-Andermann, El-Amraoui et al, EMBO J, 2000).  We will report work in progress towards describing Vezatin expression pattern in both very early embryonic stages and during the formation of EBs. In parallel, an approach using gene targeting and/or conditional gene targeting is being

POSTER 94 - MOLECULAR AND GENETIC ANALYSIS OF THE EPIDERMAL GROWTH FACTOR RECEPTOR IN MURINE PLACENTAL DEVELOPMENT

Ms. Karen Strunk
University of North Carolina-Chapel Hill
Department of Genetics
11-109 Lineberger Cancer Center
Campus Box #7264
Chapel Hill
NC/27599-7264
USA

Co-Authors: Hovick M, Amann V, Threadgill DW

Institutions: University of North Carolina, Department of Genetics

Homozygous epidermal growth factor receptor (Egfr) null mutants exhibit peri-implantation to post-natal lethality, depending on the genetic background. On a 129/SvEvTAC background Egfr homozygous null embryos die at embryonic day 11.5 due to placental defects. The placentae show a decreased size compared to those from control littermates, with a reduced spongiotrophoblast layer and disorganization of the labyrinthine layer. On an outbred CD-1 background homozygous null pups also exhibit a reduced spongiotrophoblast layer, but there is partial rescue of the labyrinthine layer. To address the strain dependant variability, a scan for genetic modifiers was performed using the outbred CD-1 background. No statistically significant loci were identified in a large backcross mapping panel. In order to partition the genetic variability potentially segregating within the outbred Swiss-derived CD-1 population, we tested 10 inbred Swiss-derived strains to determine the viability of Egfr null mutants. The strains tested include ALR/LtJ, ALS/LtJ, NON/LtJ, NOD/LtJ, ICR/HaRos, SJL/J, SWR/J, FVB/NJ, APN, and APS. We reasoned that different inbred strains derived from the same Swiss population as the outbred CD-1 mice would have captured different sets of modifier alleles. As predicted, a significant variation in the penetrance of Egfr null viability was observed in the various inbred strains.

POSTER 95 - GLOBAL DYSREGULATION OF NORMAL GENE EXPRESSION PATTERNS IN PLACENTAS OF CLONED MICE

Dr Hiroshi Suemizu
Central Institute for Experimental Animals
1430 Miyamae, Nogawa
Kawasaki
216-0001
Japan

Co-Authors: 1) Shimozawa N, 1) Tamaoki  N, 2) Aiba K, 2) Ko SHM

Institutions: 1) Central Institute for Experimental Animals  2) Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, NIH

Increased placental and birth weights are the most common abnormalities of cloned animals generated by the transfer of somatic nuclei. It has been speculated that this phenotype, called 'large offspring syndrome,' is caused by the dysregulation of control of gene expression. However, investigations have thus far been limited to the analyses of several imprinted genes and the methylation status of genomic DNA. To gain further perspective on the extent of changes, we have carried out gene expression profiling on cloned mouse placentas using the NIA 15K mouse cDNA microarray. Here we report four principal features: (1) inappropriate expression of imprinted genes, including a ~10-fold reduction of H19 levels; (2) overexpression of genes involved in placental growth; (3) increased expression of oncogenes and growth promoting genes; and (4) alteration of regulatory proteins involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase. Our results indicate that placentomegaly in cloned animals is caused by large-scale dysregulation of normal gene expression as well as the dysregulation of imprinted genes.

POSTER 96 - MOLECULAR ANALYSIS OF NONDISJUNCTION IN MICE HETEROZYGOUS FOR A ROBERTSONIAN TRANSLOCATION

Ms. Lara Underkoffler
Children's Hospital of Philadelphia
1004 ARC
34th St. and Civic Cntr. Blvd.
Philadelphia
19104
USA

Co-Authors: 1) Mitchell,LE, 2) Oakey,RJ

Institutions: 1)University of Pennsylvania School of Medicine, Department of Biostatistics and Epidemiology, 2)Children's Hospital of Philadelphia, Genetics Division, Department of Pediatrics

A Robertsonian translocation results in a metacentric chromosome from the fusion of two acrocentric chromosomes.  The gametes of Rb heterozygote mice frequently undergo nondisjunction to produce aneuploid embryos providing a good model for studying nondisjunction in mammals.  We intercrossed mice heterozygous for a (7.18) Robertsonian translocation and performed molecular genotyping of 1,927 embryos from 363 litters of mice with known parental origin, strain and age.  Nondisjunction events were scored in these mice and we then examined factors that influence the frequency of nondisjunction involving chromosomes 7 and 18.  This analysis allowed us to make a number of conclusions.  (1) The frequency of nondisjunction among 1,782 embryos, (3,564) meioses, was 16.5%.  (2) Nondisjunction events are distributed non-randomly among progeny.  This was inferred from the distribution of the frequency of trisomics and uniparental disomics (UPDs) among all the litters.  (3) Strain background did not play an appreciable role in nondisjunction frequency.  (4) There was no evidence to show an effect of maternal or paternal age on the frequency of nondisjunction.  (5) In males, but not females, the frequency of nondisjunction for chromosome 18 was significantly higher than for chromosome 7.  (6) In females the frequency of nondisjunction for chr 7 was significantly higher than for males, but there was no difference in chr 18 nondisjunction frequency between sexes.  These results show that molecular genotyping provides a valuable tool in understanding factors influencing nondisjunction in mammals.

POSTER 97 - GENETIC ANALYSIS OF A MOUSE MUTANT, X-LINKED POLYDACTYLY (XPL)

Ms Yukari Yada
Mammal. Genet., Natl. Inst. Genet.
1111 Yata
Mishima
411-8540
Japan

Co-Authors: 1) Makino S, 2) Ishiwa S. 1) Shiroishi T

Institutions: 1)Mammal. Genet., Natl. Inst. Genet.  2)Ochanomizu Univ. 

X-linked polydactyly (Xpl) is a spontaneous mouse mutation that exhibits preaxial polydactyly only on the hindfeet. To study the molecular basis underlying Xpl phenotype, we analyzed the expression patterns of several marker genes in the limb buds of Xpl embryos by in situ hybridization. At E11.5, ectopic expression of Shh, Fgf4, ptc, Gre and Hoxd11 were observed at the anterior margin of the hindlimb buds. These markers were not detected before the expression of Shh gene at the posterior margin of the limb buds. However, at E10.5, Gli, which is a downstream gene of Shh, was expressed at the anterior side of the Xpl hindlimb prior to the ectopic Shh expression. All results suggested that, in the normal limb development, Xpl gene likely acts in the downstream pathway of Shh signaling cascade.

To isolate the Xpl gene, we carried out linkage analysis of Xpl in crosses with wild mice-derived strains. As a result, Xpl was mapped to a 0.83cM interval between the microsatellite markers, DXMgc39 and DXMit32. Since in the human syntenic region, Xp22, a congenital face and limb deformity, Oral-facial-digital syndrome type 1 (OFD1: OMIM311200), has been mapped, Xpl has been thought to be a mouse model for OFD1. We mapped a murine homologue for OFD1, cXorf5, onto our map, and we excluded it from the critical region of Xpl.