ABSTRACT BOOK

Pathology of Genetically-Engineered Mice (GEM)

(So you've got a new GEM, what do you do next?)

Acknowledgements

Sponsors - The National Cancer Institute and The National Institute of Environmental Health Sciences

Organizer - Jerrold M. Ward, NCI

Organizing Committee - Jerrold M. Ward, NCI; Joel F. Mahler, NIEHS; Robert R. Maronpot, NIEHS; John P. Sundberg, The Jackson Laboratory; H. Rick Bedigian, NCI-FCRDC; Miriam Anver, NCI-FCRDC; Lino Tessarollo, NCI-FCRDC; Colin Stewart, NCI-FCRDC; Glenn Merlino, NCI; Michael Eckhaus, VRP, NIH; Timothy O'Neill, AFIP; Tracie E. Bunton, The Johns Hopkins University

Book editors - J.M. Ward, J.F. Mahler, R.R. Maronpot, J.P. Sundberg

Cover page art - Taheresa Mae Plona, NCI-FCRDC Animal Health Diagnostic Laboratory

Graphics - Richard Frederickson, Alan Kane, Ellen Frazier, NCI-FCRDC Publications Department

Conference Coordinator and staff- Margaret Fanning, NCI-FCRDC, Susan Wester, Doris Hodge

Conference Support - Patricia Brown, V.M.D., M.S., Michelle Gottholm

NIH Natcher Center - Dawn Higgs and staff

Trans-NIH Mouse Initiative http://www.nih.gov/science/mouse/
The Jackson Laboratory Mouse Genome Informatics http://www.informatics.jax.org/
T base http://tbase.jax.org/
NetVet Rodents http://netvet.wustl.edu/rodents.htm
NCI Veterinary Pathology http://www.ncifcrf.gov/vetpath/
BiomedNet (Knockout mouse database) http://www.biomednet.com/
The Whole Mouse Catalog http://www.rodentia.com/wmc/

Faccini, J.M., Abbott, D.P. and Paulus, G.J.J., 1990. Mouse Histopathology: A Glossary For Use in Toxicity and Carcinogenicity Studies. Amsterdam, Elsevier.

Frith, C.H. and Ward, J.M., 1988. Color Atlas of Neoplastic and Non-neoplastic Lesions in Aging Mice. Amsterdam: Elsevier, pp.109, 354 color figures,
Green, E.L. (ed.) 1966. Biology of the Laboratory Mouse. New York: dover, pp.706.
Jones, TC (editor). 1983-98 Monographs on Pathology of Laboratory Animals. Series Volumes. Washington DC: ILSI Press and Springer Verlag, http://www.ilsi.org/animal.html

Lyon M.F., Rastan S. and Brown S.D.M., 1996. Genetic Variants and Strains of the Laboratory Mouse. Third edition, Volumes 1 and 2. Oxford: Oxford University Press.

Maronpot, RR (editor), Boorman GA and Gaul BW (co-editors). 1999. Pathology of the Mouse: Reference and Atlas. Vienna, IL: Cache River press, In press.

Mohr U, Dungworth DL, Ward JM, Capen CC, Carlton W, and Sundberg JP, 1996. Pathobiology of the Aging Mouse, Volumes 1 and 2. Washington DC: ILSI Press. http://www.ilsi.org/animal.html

Percy D.H. and Barthold S.W. 1993. Pathology of Laboratory Rodents and Rabbits. Ames: Iowa state University Press.

Rugh, R. The Mouse: Its Reproduction and Development. Minneapolis: Burgess. 1968.

Turusov,V. and Mohr, U., editors. 1994. Pathology of Tumours in Laboratory Animals, Volume II- Tumours of the Mouse. Lyon, IARC Scientific Publications.

Development http://www.biologists.com/Development/
Genes & Development http://www.genesdev.org/
Transgenics (Harwood Academic Publishers) http://www.gbhap-us.com/journals/385/385-top.htm
Trends in Genetics http://www.elsevier.nl/inca/publications/store/4/0/5/9/1/8/
Toxicologic Pathology (STP) http://www.toxpath.org/
Veterinary Pathology (ACVP) http://vetpathol.cvm.uiuc.edu/vetpathol
Laboratory Animal Science (AALAS) http://www.aalas.org/pubs.htm
The American Journal of Pathology http://www.edoc.com/PATHOLOGY/
Laboratory Investigation http://info.med.yale.edu/labinvest/welcome.html

Day 1 Wednesday, February 24, 1999

Introduction and Resources

Moderator: Dr. JM Ward, NCI

8:30am Welcome and Introduction to Symposium- JM Ward, NCI

8:45am Mutant Resources - Role of a Repository, John Sharp, The Jackson Laboratory
9:15am Approaches for Cryopreserving Genetically Engineered Mice, Hendrick G. Bedigian, NCI-FCRDC

Techniques for the detection and analysis of normal and inappropriate gene expression.

Moderator: Dr. Maria Lucia Dagli, IARC and University of Sao Paulo

9:30am Genetic Events Detected By In Situ Hybridization (ISH) - Cecil Fox, Molecular Histology Labs.

10:00am Break

10:30am Immunohistochemistry: Methods and Troubleshooting - Craig Pow, Vector/Novocastra Labs.

11:00am Use of Confocal Microscopy Techniques In The Study of Transgenic and Knockout Mouse Genetics-
James Resau, NCI-FCRDC

11:20am Laser Capture Microdissection: Molecular Analysis of Tissues, Robert F. Bonner, NICHD

11:45am Molecular Pathology Techniques for Genetically Engineered Mice:
Overview of Applications - Glenn Merlino, NCI

12:15pm Lunch

Techniques for analyzing mouse tissues

Moderator: Robert Maronpot, NIEHS

1:15pm Measurement of Cell Replication and Apoptosis in Mice, Robert R. Maronpot, NIEHS

1:45pm Analysis of Mammary Tissue in Transgenic and Knock-out Mouse Models, Lothar Hennighausen, NIAMS

2:15pm Correlation of Histopathologic and Genetic Changes During Mammary Tumor Progression in
C3(1)/TagTransgenic Mice, Jeff Green and Masa Shibata, NCI

2:45pm Plain Radiography of Mice, M. Mankani, NIDCR

3:00 Break

3:20pm Pathology Evaluation Of Genetically-engineered Mice, Charles Montgomery, Baylor College of Medicine

Strain- and Age-related Pathology and Some Tumors

Moderator: Tim O'Neill, AFIP

4:00pm Effects Of Genetic Background On The Phenotype of Induced Mutants, Joel Mahler, NIEHS

4:30pm Pathology of GEM Mouse Lines Used in New Short-term Carcinogenesis Bioassays, Peter Mann, EPL

5:00pm Lymphomas In Mice, Lekidelu Taddesse-Heath, NIAID

5:30pm Unique Proliferative Lesions in Genetically-engineered Mice, Miriam Anver, NCI-FCRDC

Day 2 Thursday, February 25, 1999

Biology and Pathology of Normal and Abnormal Embryonic Development

Moderator: Dr. Colin Stewart

8:30am The Development And Experimental Manipulation Of The Early Mouse Embryo,
Colin Stewart, NCI-FCRDC

9:00am Methods For Handling Mouse Embryos For Anatomy Studies: Gross and Microscopic Anatomy of the
Mouse Embryo, Matt Kaufman, Edinburgh U.

9:30am Extra-embryonic Embryonic Tissues of the Mouse, Jerrold M. Ward, NCI

10:00am Break

10:30am Analysis of Skeletal Patterning and Morphogenesis in Mouse Embryos, Susan Mackem, NCI

11:00am Normal Brain Development And Gene Discovery, David Jacobowitz, NIMH

11:30am Gene Targeting of Neurotrophins and Their Receptors: Pleiotropic Developmental Defects,
Lino Tessarollo, NCI-FCRDC

12:00am Strategies for Behavioral Phenotyping of Transgenic and Knockout Mice, Jacqueline N. Crawley, NIMH

12:30pm Lunch

1:30pm Mouse Imaging. R. Nick Bryan, NIH Clinical Center

Special Pathology of GEM

Moderator: John Sundberg, The Jackson Laboratory

1:45pm Skin Pathology of Spontaneous, Transgenic, and Targeted Mouse Mutations, John P. Sundberg, The
Jackson Laboratory

2:15pm Techniques For The Evaluation of Neuropathology In Mice, Roderick T. Bronson, Tufts U.

2:45pm A Neurological Syndrome in FVB/NCr Mice. Jerrold M. Ward, NCI

3:00pm Pathology of Transgenic Mice With Abnormal Lipid Metabolism, Marion J.J. Gijbels, Leiden University
Medical Center, The Netherlands

3:15pm Break

Moderator: Tracie E. Bunton, The Johns Hopkins University

3:45pm Interpretation of Ocular Pathology in Genetically-engineered Mice And Other Mutant Mice,
Richard S. Smith, The Jackson Laboratory

4:15pm Cardiovascular Pathology of Genetically-engineered Mice, Tracie E. Bunton, The Johns Hopkins University

4:45pm Renal Diseases in Genetically-Engineered Mice, Michael Eckhaus, NIH

5:15pm Inflammatory Bowel Disease In Mouse Models: Role of Intestinal Microbiota As Proinflammatory
Modulators, James G. Fox, MIT

5:45pm Symposium Summary, Jerrold M Ward, NCI

Abstracts

Mutant Resources - Role of a Repository

John J. Sharp Ph.D.
The Jackson Laboratory, Bar Harbor Me. 04609
jjs@aretha.jax.org, 207-288-6211, Fax 207 288-6149

Genetically defined mice have been widely utilized to further our understanding of mammalian biology. They have provided model systems for analyzing the defects in comparable human disorders and have also been widely used for preclinical and toxicological testing of therapeutic agents. Of particular importance, the genetics of the mouse is more widely characterized and understood than for any other experimental animal. Inbred strains, strains carrying spontaneous mutations or chromosomal aberrations, recombinant inbred strains, and congenic inbred strains have all contributed to this understanding (1).
The ability to selectively alter the mouse genome through gene transfer (transgenic mice) (2), homologous recombination (gene targeting) (3), insertional mutagenesis (4) and chemical mutagenesis (i.e. 5) is providing new and powerful tools for biomedical research. The production and use of transgenic mice has expanded greatly since its inception in 1980 and gene targeting technology is developing even more rapidly. The generation of conditional mutants (6) promises to further expand the utility of these strains.

In order to insure the maximum use of these new mouse models by the scientific community, a support system consisting of genetic resources and informatics resources has been established by the NIH. The Induced Mutant Resource (IMR) at The Jackson Laboratory serves as the national repository for genetically engineered mouse strains (7). The major functions of the IMR are to identify, import (establish a health standard), cryopreserve gametes or embryos, make new models, provide information, and distribute genetically engineered strains of mice. A similar, but different, repository has been established in Europe. The increasing production, utility and demand for these strains will require additional repositories in the near future.

Repositories for genetically engineered mouse strains are faced with unique breeding, health maintenance, genetic quality control and legal considerations that are not often encountered with inbred strains. Also, because one function of a repository is to transfer mutations onto different genetic backgrounds (make congenic strains) the effect on the phenotype of the genetic background must be evaluated. These issues will be discussed.

1. Davisson MT, Sharp JJ. 1998. Repositories of Mouse Mutations and Inbred, Congenic, and Recombinant Inbred Strains. In: Systematic Approach to Evaluation of Mouse Mutations, Sundberg JP and Boggess D (eds.), CRC Press, (in press).
2. Capecchi MR. 1989. Altering the genome by homologous recombination. Science, 244: 1288-92.
3. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A, 77: 7380-4.
4. Friedrich G, Soriano P. 1993. Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells. Methods Enzymol, 225: 681-701.
5. Schimenti J, Bucan M. 1998. Functional genomics in the mouse: phenotype-based mutagenesis screens. Genome Research, in press.
6. Kuhn R, Schwenk F, Aguet M, Rajewsky K. 1995. Inducible gene targeting in mice. Science, 269:1427-9.
7. Sharp JJ, Davisson, MT. 1994. The Jackson Laboratory Induced Mutant Resource.. Lab Animal. 23:32-40.
Internet Sources
The Jackson Laboratory Induced Mutant Resource, http://lena.jax.org/resources/documents/imr/
NIH Animal Genetic Resource http://www.nih.gov/od/ors/dirs/vrp/nihagr.htm
Taconic Animal Models http://www.taconic.com/anmodels/animlmod.htm
National Academy Press - Biological Models http://www.nap.edu/readingroom/enter2.cgi?0309060354.html

Approaches for Cryopreserving Genetically Engineered Mice
H. G. Bedigian Ph.D. SAIC-FCRDC Frederick, MD 301
bedigianh@mail.ncifcrf.gov, 663-4152, Fax 301 846-6165

L. Mobraaten Ph.D. The Jackson Laboratory Bar Harbor ME
lem@aretha.jax.org, 207-288-66373

The ability to alter mammalian genomes opened an exciting new era of research into the genetic bases of human health and disease. The technology that allows us to selectively alter mammalian genomes has led to a rapidly expanding production of transgenic, targeted mutant and chemically mutagenized rodents and thus necessitates the development of new concepts in animal colony management. The limited physical space to maintain colonies and the risk of losing mice make cryopreservation a valuable and cost effective means to maintain strains whose current use is limited or non-existent. Strain cryopreservation also provides insurance against the loss of a colony due to disease or to genetic and environmental factors. Recent achievements in cryopreserving mouse oocytes, ovaries, and sperm, in addition to embryos, increases the effectiveness of a cryopreservation bank in having germplasm from some of the unique animals that otherwise could be lost.

Genetic Events Detected by in situ Hybridization

Cecil H. Fox, Ph.D.
Molecular Histology, inc.
18536 Office Park Drive, Montgomery Village, MD 20879
301-216-1564, jwgibbs@us.net, www.us.net/mol.hist

In situ hybridization is a technique established by Gall and his students (1969) initially for localization of genes in cytogenetic studies. Subsequently, the technique has found many other uses and when properly done can yield reliable information about expression of mRNA, viral RNA, the presence of DNA and its location2. In working with GEM, the technique is usually applied to expression (or lack thereof) of mRNA. Selection of in situ techniques depends on the specimen, the experiment and the expectations of the investigator. In mouse studies, two techniques have been particularly useful, whole embryo nonradiolabeled probes and histological study of cells or tissues.
While either of these may reveal important results, combined, they may support each other in this technically demanding research technique. The principle of whole embryo studies is that chromogenic probes are hybridized with mRNA produced by organs or in organogenesis. Since an entire expressing organ may be detected in a tissue layer hundreds of cells thick, the image is amplified by the numbers of cells to give a clear indication of where genes are being expressed in the whole embryo.
When whole mount in situ fails to show gene expression the causes may be due to complex variables such as expression in limited localities too small or hidden to be seen. In such instances, sectioning of the tissue may be the next recourse. Frozen sections of embryos can sometimes be used despite the morphological problems of cryotomy and the difficulties of using stringent washes with subsequent tissue loss. We have used paraffin embedded, formaldehyde fixed tissues as a course of first resort. Tissue sections are mounted on slides coated with 3-aminopropyltriethoxysilane and handled with strict precautions against RNAase contamination.
The next major decision is whether to label probes with chromogenic markers or with radioisotopes3. Because we want the highest sensitivity and we know that isotopes are more sensitive than colorimetric reagents, all of our probes are made from cDNA templates with SP6, T7, or T3 polymerases and incorporate 35S, 33P, 3H, or 125I nucleotide triphosphates. Radiolabeled areas are detected by autoradiography on emulsion, or more recently by phosphorimaging4 that has the added
advantage of being quantitative.
With colorimetric reagents, nucleotides may be labeled with BuDR, fluorescein, digoxygenin and biotin among others. Colorimetric detection is usually with a secondary antibody with alkaline phosphotase, horse-radish peroxidase, or beta galactosidase and detected with DAB, BPIC-NBT, X-gal, immunogold or other reporter reagents. Following the hydridization, the slides my be photographed either with a microscope or macroscope5 or in the case of imaged slides, software is
available for further analysis.
If these techniques seem tedious and labor intensive it is because they are. Inattention to details, substitution of reagents, technical deviations will all be rewarded by either no results or inexplicable and unreliable ones. Unrealistic expectations include using oligomer or PCR derived probes to detect low levels of expression. There is a previously unwritten law that states, "success in in situ hybridization is a function of the length of probe sequences in relation to the numbers of copies to be detected." For example, with 10kb of probe sequence divided into 200 base units we can detect as few as 15 copies per cell.
Use of in situ methods can answer a number of important issues in altered mouse studies.
1. What organs (or cells) express what genes in development or adults?
2. Are transgenic animals functional in the desired way?
3. Are knockouts really knockouts?
4. Does expression in one organ influence expression in others?
5. What are the limits of gene expression?
6. Are negative results also results?

1. Gall, J. G. and M. L. Pardue. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Genetics 63:378-383, 1969
2. Leitch, A. R. T. Schwarzacher, D. Jackson and I. J. Leitch, In Situ Hybridization, Royal Microscopical Society, Microscopical Handbooks 27. 1994, London. or Choo, K.H.A Methods in
Molecular Biology, 33, In Situ Hybridization Protocols, Humana Press, Totowa, NJ 1994.
3. Fox CH, Cottler-Fox M. In situ hybridization for the detection of HIV RNA in cells and tissues. In: Current Protocols in Immunology (Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W eds), Wiley, NY, 1993.
4. Fox, C. H., Hoover, S., Currall, V. R., Bahre, H. J., and Cottler-Fox, M. HIV in Infected Lymph Nodes. Nature 370: 256, 1994.
5. Fox CH, Dreyfuss R. Photography and molecular biology: in situ hybridization, autoradiography and immunogold. J Biol Photog 1992, 60(1): 39-44.

Immunohistochemistry: Methods and Troubleshooting

Craig Pow, Ph.D.
Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010, U.S.A.
(650) 697-3600, FAX: (650) 697-0339
e-mail: vector@vectorlabs.com, http://WWW.vectorlabs.COM

Immunohistochemistry (IHC) provides information about a given antigen¹s presence at the tissue and cellular levels. The extent of it¹s distribution in the tissue, and it¹s precise location within a cell can be elucidated through observation of specific staining. Careful interpretation of these staining patterns before (control tissue), and after treatment (test tissue), or during proliferation and differentiation, can provide valuable data about a protein¹s regulation and possible function.

For IHC to be effective, the method used must be specific, sensitive and produce no or little background. The most widely used IHC systems utilize the enzymes peroxidase or alkaline phosphatase. These enzymes are coupled to detection reagents which identify the primary antibody bound to the target antigen, amplify the signal, and finally produce a colored precipitate on the section.

In spite of using the most established IHC methods, the tissue characteristics may ultimately determine the success of the application. Optimal antigen preservation and morphology are crucial for obtaining consistent, reliable and meaningful results. To achieve this, parameters including tissue collection, handling and preparation, fixatives and fixation times, temperature, sectioning and storage must be considered. The detection procedure itself may have to be modified depending on the tissue available, how it was prepared, and the antibody intended for use. The origin and composition of the tissue also may dictate what procedures are required to get the best results. Defined controls are essential in each experiment for correct evaluation and validation of positive tissue staining.

Tissue of mouse origin is a special case, since mouse models are widely used and the majority of monoclonal primary antibodies are developed in mice. The use of anti-mouse secondary antibodies to detect mouse primary antibodies on mouse tissue is problematic. However, through the use of novel methods, these concerns can be overcome, and thus, opens up the possibility of using monoclonal mouse primary antibodies in mouse transgenic, knockout and xenograft studies.

1) Polak, J.M. and Van Noorden, S. (1997) Introduction to immunocytochemistry. 2nd Ed. Microscopy Handbook Series-37, Springer-Verlag, New York, NY.

2) Analytical Morphology: Theory, Applications and Protocols. Gu, J. (ed) 1997. Eaton Publishing Co., Natick, MA.

Use of Confocal Microscopy Techniques in the study of transgenic and knockout mouse genetics.

James H. Resau, Ph.D.
Director, Analytic Cellular and Molecular Microscopy Laboratory
NCI-FCRDC, ABL-BRP, Building 538, Room 157
Frederick, MD 21702-1201

Resau@mail.ncifcrf.gov Telephone 301-846-1554

Transgenic and knockout mice have known genetic modulations that result in objectively identifiable and measurable phenotypic changes. Microscopic evaluations of a "changed morphology" are reproducible within an individual or a group on the basis of qualitative evaluations. Quantification is ordinarily not transferable and is usually expressed suggestively as gradations of 0 to ++++. Confocal microscopy changes that paradigm and allows not only high resolution light microscopy analysis of cells and tissues but provides objective, measurable quantification of protein expression.

Confocal microscopy utilizes laser light, high numerical aperture lenses and digital image recording of specimens using a known pin hole aperture that determines volume. These microscopes are computer controlled with stored parameter files that insure reproducible application of laser power, pin hole aperture, diachronic / filter set alignments, etc so that imaged specimens are "equal". The pinhole setting determines the volume of the sample imaged. The use of parameter files combined with the inherent proportionality of immunostaining dilutions allows one to not only precisely determine within a specimen the location and expression of a protein but to also quantitative that expression in either relative or absolute terms using pixel intensity and common image analytical methods.

Confocal microscopes allow one to image either sequentially or simultaneously depending on laser and filter selections, multiple fluorophore as well as DIC images of cells or sections. One can then easily over-lay the imagery and asses co-localization aspects of antigens or proteins of interest. Examples of the expression of such methods in mouse tissues are presented. High-resolution images of the mouse eye expression of microphthalmia gene (mi), linked to Waarenburg syndrome, can result in mutation. The mi was imaged using green, tubulin with Rhodamine, DNA using Dapi and eye histology using DIC or Nomarski optics. In wild type mice the mi localizes to the nucleus. But mutant mice have expression of mi in both the nucleus and the cytoplasm of their RPE cells. The localization, co-localization and quantification of protein amounts are all possible and easy using confocal microscopic techniques.

Newer confocal systems that take advantage of 2-photon emission and high-resolution optics will even further enhance the qualitative and quantitative expression of genetic events in transgenic and knockout phenotypes.

Klineberg, E., Tsarfaty, I., Alvord, W., Sathyanarayana, B., Resau, J; Correction and quantification of normal differentiation in human epithelium: Application for Optimas 4.0 image analysis program. Cell Vision 3:402-6, 1996.

Minc-Golomb, D., Yadid, G., Tsarfaty, I., Resau, J., Schwartz, J., In vivo expression of inducible nitric oxide synthase in cerebellar neurons. J Neurochem 66:1504-9, 1996.

Tsarfaty, I., Rong, S., Resau, J., Shen, R., Pinto da Silva, P., Vande Woude, G; Met signaling in mesenchymal to
Epithelial conversion. Science 263:98-101, 1994.

Vande Woude, G., Jeffers, M., Cortner, J., Alvord, W., Tsarfaty, I., Resau, J., Met-HGF/SF: Tumorigenesis,
invasion, and metastases. Plasminogen Related Growth Factors, Proceedings of the CIBA Symposium 212:119-132, 1997.

Laser Capture Microdissection : Molecular Analysis of Tissue

Robert F Bonner, Ph.D.

Chief, Section on Medical Biophysics, Laboratory of Integrative and Medical Biophysics, NICHD, Bldg 13 Rm 3N17 [Lab Bldg 37 Rm 2B13], NIH, Bethesda, MD 20892-5766, Phone: 301-435-1946, Fax: 301-496-6608, Email: bonner@helix.nih.gov, http://dir.nichd.nih.gov/lcm/lcm.htm

As basic research compiles an expanding list of expressed human genes, a major scientific and medical challenge is the application of this knowledge to understand the molecular events which drive normal tissue morphogenesis and the progression of pathologic lesions in actual tissue. With the advent of PCR, quantitative RT-PCR, and microhybridization arrays, it now becomes feasible to extract DNA or RNA from a thousand cells or less and analyze a parallel panel of hundreds or even thousands of genetic markers. Since cells in complex tissue are biochemically and physically affected by surrounding cells and by remote stimuli from greater distances, the task of analyzing critical gene expression patterns in development, normal function, and disease progression is critically dependent on the extraction of specific cells from their complex tissue milieu. We have developed Laser Capture Microdissection to provide a rapid, reliable method of procuring pure populations of specified cells from specific microscopic regions of tissue sections for subsequent quantitative, multiplex molecular analysis. Previous manual microdissection techniques have demonstrated the ability to target and isolate specific populations of cells from stained tissue sections and amplify the procured material using PCR techniques. LCM technology (now commercially developed through a CRADA with the NIH) is an easy to use technique with the ability to target and accumulate individual cells from a tissue and has been adapted for a variety of molecular analysis techniques such as cDNA library construction, PCR of DNA alterations, QRT-PCR and array hybridization for determined of the patterns of gene expression, and a variety of protein analysis methods. The advantages of the LCM system include 1) one step transfer of the cells of interest from the tissue section on the glass slide to the polymer film. 2) Since only the targeted cells are affected by the process, multiple subpopulations of cells can be serially isolated from the same section in order to compare spatial variation in patterns of multiple gene expression of similar or dissimilar but interacting cells in a complex tissue. 3) No micromanipulation is required to perform LCM. 4) The exact morphology of the procured cells is retained and held on the transfer film; thus constituting a diagnostic record of the exact cells undergoing molecular analysis.

LCM websites: http://dir.nichd.nih.gov/lcm/lcm.htm
http://www.arctur.com

Bonner RF, Emmert-Buck M, Cole K, Pohida T, Chuaqui R, Goldstein S, Liotta LA. 1997. Laser capture microdissection: molecular analysis of tissue. Science 278:1481,1483.
Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. 1996. Laser capture microdissection. Science 274:998-1001.

Luo L, Salunga RC, Guo H, Bittner A, Joy KC, Galindo JE, Xiao H, Rogers KE, Wan JS, Jackson MR, Erlander MG. 1999. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat Med 1999 5:117-22.
Suarez-Quian, et al., Biotechniques, Feb 99, in press

Molecular Pathology Techniques for Genetically-Engineered Mice: Overview of Applications

Glenn Merlino, Ph.D.
Laboratory of Molecular Biology, National Cancer Institute, Building 37,
Room 2E24, Bethesda, MD 20892-4255
gmerlino@helix.nih.gov

The advent of technology permitting precise genetic manipulation of murine embryos has revolutionized the mouse as a model system, while providing seminal clues to the in vivo functions of countless gene products. Through embryonic pronuclear microinjection, expression of wild type or mutant proteins, encoded within transgenes, can be targeted to any of a number of ectopic sites in embryos and/or adult tissues, resulting in a genetic gain-of-function. Conversely, genetic loss-of-function can be achieved through targeted inactivation of designated genes by homologous recombination in embryonic stem cells, which can be reconstituted into a so-called knockout mouse. Recently, impressive variations on these two basic genetic themes have been introduced that allow additional control over, and fine-tuning of, functional modifications. These various genetic advancements often result in unpredictable and novel phenotypes, providing veterinary pathologists with new opportunities and challenges. I will review the technological possibilities in this brave new world through the presentation of specific experimental examples, and by illustrating how the resulting novel mouse models can be analytically dissected. The following references exhibit an array of molecular pathology techniques that can be used in the analysis of genetically engineered mice:

1. Celli, G., LaRochelle, W.J., Mackem, S., Sharp, R., and Merlino, G. 1998. Soluble dominant negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning. The EMBO J. 17: 1642-1655.

2. Takayama, H., LaRochelle, W.J., Anver, M., Bockman, D.E., and Merlino, G. 1996. Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development. Proc. Natl. Acad. Sci. U.S.A. 93: 5866-5871.

3. Sharp, R., Babyatsky, M.W., Takagi, H., Tågerud, S., Wang, T.C., Bockman, D.E., Brand, S.J., and Merlino, G. 1995. Transforming growth factor alpha disrupts the normal program of cellular differentiation in the gastric mucosa of transgenic mice. Development 121: 149-161.

Measurement of Cell Replication and Apoptosis in Mice
Robert R. Maronpot, D.V.M., M.S., M. P.H.Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709

In the last 10 years, appreciation of the complexities of the cell cycle and the identification of numerous positive and negative molecules that control cell proliferation and cell death (apoptosis) has prompted the development of many types of genetically engineered mice for study of growth, development, and carcinogenesis. At some stage in the utilization of these genetically engineered mice, a need arises for practical estimation of cell proliferation and cell death in target tissues of interest. The first critical consideration in this regard relates to study design, and that will be influenced by the question(s) being asked, the target tissue of interest, and the expected magnitude of the response. Because of the labor intensive nature of cell proliferation and apoptosis measurements, it is recommended that all aspects of the study be discussed with collaborating pathologists knowledgeable in histomorphology prior to settling on an appropriate study design. Different target tissues have different intrinsic rates of cell proliferation, and duration of labeling must account for these intrinsic rates. Decisions regarding using exogenous markers such as tritiated thymidine or bromodeoxyuridine versus using endogenous markers such as proliferating cell nuclear antigen, mitoses, or histone mRNA will influence study design. Design considerations must take into account: the duration of labeling (pulse versus continuous); normal circadian cycles; the method of tissue preparation (cell suspensions for flow cytometry versus tissue sections for immunohistochemistry or autoradiography); the method and duration of tissue fixation; whether or not to use surgically implanted osmotic minipumps; how the tissue or organ counts will be evaluated (random fields or specific subanatomic regions or cell types); how many cells will need to be evaluated to generate appropriate measurements; whether measurements will be made by computer assisted/automated procedures or by brute force; and based upon expected parameter variability, how many animals will be necessary for each time point. Once a study is completed and data are generated, important factors related to interpretation of results become of paramount importance. Here the input from the pathologist is again critical as the cell proliferation and cell death metrics need to be considered in light of changes in organ size and weight and the presence of histopathologic lesions in target tissues and relevant related tissues. Interpretation will undoubtedly need to be made in light of tissue pathologies and in consideration of the balance between cell proliferation and cell death. This presentation will focus on design and methodology issues that are important in generating reliable cell proliferation and apoptosis data.

1. Christensen JG, Gonzales AJ, Cattley RC, and Goldsworthy TL. Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens. Cell Growth and Differentiation (in press), 1998.
2. Eldridge SR, Tilbury LF, Goldsworthy TL, and Butterworth BE. Measurement of cehmically-induced cell proliferation in rodent liver and kidney: a comparison of 5-bromo-2'-deoxyuuridine and 3H-thymidine administered by injection or osmotic pump. Carcinogenesis 1991 11:2245-2251.
3. Enomoto A, Sandgren EP, Maronpot RR. Interactive effects of c-myc and transforming growth factor alpha transgenes on liver tumor development in simian virus 40 T antigen transgenic mice. Vet Pathol 1998 Jul;35(4):283-91
4. Foley J, Ton T, Maronpot R, Butterworth B, Goldsworthy TL. Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ Health Perspect 1993 Dec;101 Suppl 5:199-205
5. Fransson-Steen R, Goldsworthy TL, Kedderis GL, Maronpot RR. Furan-induced liver cell proliferation and apoptosis in female B6C3F1 mice. Toxicology 1997 Mar 28;118(2-3):195-204
6. Goldsworthy TL, Fransson-Steen R, and Maronpot RR. Importance of and approaches to quantitation of hepatocyte apoptosis. Toxicol. Pathol. 1996 24:24-35.
7. Goldsworthy, TL, Connolly R, and Fransson-Steen R. Apoptosis and cancer risk assessment. Mutat. Res. 1996 365:71-90.
8. Greenwell A, Foley JF, Maronpot RR. Detecting proliferating cell nuclear antigen in archival rodent tissues. Environ Health Perspect 1993 Dec;101 Suppl 5:207-9
9. Nyska A, Maronpot RR, Eldridge SR, Haseman JK, Hailey JR. Alteration in cell kinetics in control B6C3F1 mice infected with Helicobacter hepaticus. Toxicol Pathol 1997 Nov-Dec;25(6):591-6
10. Ton, TT, Foley, JF, Flagler, ND, Gaul, BW and Maronpot, RR. Feasibility of administering 5-bromo-2'-deoxyuridine (BRDU) in drinking water for labeling S-phase hepatocytes in rats and mice. Toxicol. Methods 1977 7: 123-136.

Lothar Hennighausen and Gertraud W. Robinson
Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, Building 8, Room 101, National Institutes of Health, Bethesda, Maryland 20892
tel. 301-496-2716, FAX 301-480-7312, e-mail hennighause@nih.gov, http://mammary.nih.gov/lgp

Genes and signaling pathways controlling normal mammary gland development and neoplastic transformation are frequently studied in mice in which genes are ectopically expressed (transgenic) or deleted from the genome (knock-out). In order to evaluate the effects of the transgene or the gene deletion, mammary tissue has to be subjected to functional, histochemical and molecular tests. In general, various parameters in glandular structure and physiology can be affected at several distinct stages of development. Alterations can be seen in the embryonic anlage, in the immature virgin, during puberty, pregnancy, and lactation and in the process of remodeling during involution. A series of tests will be described that allow the investigator to evaluate the physiological state of the mammary gland in the mouse model under investigation. Detailed protocols will be available at http://mammary.nih.gov/tools/

Functional Tests
1. Evaluate whether mice can lactate.
2. Evaluate the amount of milk produced as indicated by the weight gain of pups.

Histochemical Tests
1. Evaluate ductal development in the immature virgin, during puberty and during pregnancy on the level of whole mounts.
2. Evaluate alveolar structures during pregnancy, lactation and involution on the level of whole mounts.
3. Evaluate ductal and alveolar architecture on histological thin sections.

Molecular Tests
1. Analyze the expression of well-characterized markers of differentiation. For each stage of mammary development specific markers have been identified and should be established on the level of RNA and protein. For example, the activity of the Jak2/Stat5 pathways reflects the differentiation of mammary epithelial cells and the expression of different milk protein genes corresponds to distinct stages of pregnancy and lactation.

References http://mammary.nih.gov/tools/

Correlation of Histopathologic and Genetic Changes During Mammary Tumor Progression in C3(1)/Tag Transgenic Mice

Masa-Aki Shibata, D.M. Sci. and Jeffrey E. Green, M.D., Transgenic Oncogenesis Group, Laboratory of Cell Regulation and Carcinogenesis , DBS, NCI, Bethesda, MD 20892
Shibata@ncifcrf.gov, 301-435-5086, Jegreen@nih.gov, 301-435-5193, fax 301-496-8395

The C3(1)/SV40 Tag transgenic model for mammary cancer shares many histopathologic similarities with human breast cancer including the development of lesions resembling human ductal carcinoma in situ (DCIS) and infiltrating ductal adenocarcinoma. Mammary tumors tend to arise from ductal epithelial cells located in the distal portions of the ductal tree close to or within the terminal end buds. Unlike other promoters commonly used for targeting the mammary epithelium in transgenic experiments, the C3(1) promoter does not require hormone stimulation for expression and is not expressed in differentiating alveolar cells in this model. Mammary lesion development in this model is highly predictable which has allowed us to attempt to identify particular genetic alterations, which may occur at different stages of mammary cancer progression. We have taken several approaches to determine what genetic changes affecting cell cycle progression and apoptosis may influence tumor progression in this model. At the early stages of lesion development, including atypical hyperplasia and nodular atypical hyperplasia, loss of expression of the cell cycle inhibitor p21 (through the Tag-induced inactivation of p53) leads to overexpression of several cyclins and cyclin-dependent kinases resulting in increased cellular proliferation. We have also demonstrated that at early stages of tumor development there is a dramatic rise in apoptosis levels during the transition from normal epithelium to mammary epithelial preneoplasia, which is associated with a significant elevation in the expression of the apoptosis-inducer, bax. The functional significance of bax expression during tumor progression in vivo has been confirmed using C3(1)/Tag transgenic mice which lack one or both wild-type alleles of bax, where increased numbers of tumors growing at an accelerated rate and larger tumor size results in significantly reduced survival rates. The bax-associated change in rates of tumor progression appears to be the result of alterations in apoptosis, specifically at the pre-neoplastic stage. The function of bax appears to be completely independent of p53 in this model of mammary oncogenesis. During late stages of tumor progression, the region of mouse chromosome six containing ki-ras is amplified during leading to elevated expression of ki-ras and MAP kinase. This amplification is functionally significant, as C3(1)/Tag mice lacking one allele of ki-ras have retarded tumor growth. The C3(1)/Tag transgenic model of mammary cancer is useful for identifying genetic alterations that may be important at different stages of tumor progression. Bax, p21, and ki-ras appear to be critical regulators of tumor progression in this model of mammary cancer.

Liu, M.L., Von Lintig, F.C., Liyanage, M., Shibata, M-A., Jorcyk, C.L., Ried, T., Boss, G.R., and Green, J.E.: Amplification of Ki-ras and MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice. Oncogene 18:2403-2411, 1998.

Maroulakou, I. G., Anver, M., Garrett, L. and Green, J. E.: Prostate and breast cancer in transgenic mice carrying a rat C3(1) SV40 TAG fusion gene. Proc. Natl. Acad. Sci. USA 91: 11236-11240, 1994.

Maroulakou, I., Shibata, M.-A., Jorcyk, C. L., Chen, X-X. and Green, J. E.: Reduced p53 dosage is associated with mammary tumor metastases in C3(1)/TAG transgenic mice. Molecular Carcinogenesis 20: 168-174, 1997.

Shibata, M.-A., Maroulakou, I. G., Jorcyk, C. L., Gold, L. G., Ward, J. M. and Green, J. E.: p53-independent apoptosis during mammary tumor progression in C3(1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from preneoplasia to carcinoma. Cancer Res. 56: 2998-3003, 1996.

Shibata, M.-A., Ward, J. M., Devor, D. E., Liu, M.-L. and Green, J. E.: Progression of prostatic intraepithelial neoplasia (PIN) to invasive carcinoma in C3(1)/Tag transgenic mice: histopathologic and molecular alterations. Cancer Res. 56: 4894-4903, 1996.

Shibata, M.-A., Liu, M.-L., Jorcyk, C.L., Yoshidome, K., Welch, V., and Green, J.E.: The C3(1)/SV40 T-antigen transgenic mouse model of prostate and mammary cancer Toxicologic Pathology, 26(1):177-182, 1998.

Yoshidome, K., Shibata, M.-A., Jorcyk, C.L., Liu, M.-L., Maroulakou, I.G., Welch, V., and Green, J.E: Genetic alterations during the development of mammary and prostate cancer in the C3(1)/Tag transgenic mouse model. International Journal of Oncology, 12:449-453, 1998.

Plain Radiography of Mice

Mahesh H Mankani M.D.
Pamela Gehron Robey Ph.D.

Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health National Institutes of Health, 9000 Rockville Pike, Building 30, Room 228, Bethesda, Maryland 20817
Phone: 301-496-1735, Facsimile: 301-402-1511, e-mail: mankani@nih.gov

Radiographic modalities available for mouse imaging include plain radiographs, CT scans, ultrasonography, and magnetic resonance imaging. Our laboratory has been exploring methods for using plain radiographs to visualize mouse hard tissues in a variety of transgenic and implantation models.

Plain radiographs offer several advantages while suffering from a variety of limitations. They are characterized by a relatively modest radiation exposure, requiring of minimal operator skill, speed, ease of use, and low cost. They can be used with contrast agents to provide images of luminal structures, whether intra-vascularly or within the gastrointestinal and genitourinary tracts. Plain radiographs in mice offer disadvantages similar to those found in clinical patients. They are appropriate for hard tissue and lung field visualization but fail to distinguish soft tissues. They offer a 2-dimensional representation of tissues while being unable to provide volumetric or cross-sectional information. They provide only static images and are inappropriate for capturing rapidly changing phenomena.

We rely on the Faxitron MX-20 Specimen Radiography System (Faxitron X-ray Corporation; Wheeling, Illinois). It's compact size, ease of operation, and ability to create magnified images with high resolution, make it appropriate for visualizing hard tissues.1 With proper orientation of the mice and digitization of the images, differences between wild type and transgenic mice can be obtained. For instance, bone length and width, degree of mineralization, trabecular pattern, and shape can be measured and compared. The spine, long bones, and skull can each be targeted. We have also used the unit to complete mouse angiograms and sialograms.2

References

1. Xu T, Bianco P, Fisher LW, Longenecker G, Smith E, et al. Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice. Nat Genet 1998; 20(1):78-82.

2. Wang S, Baum BJ, Mankani MH, Sun D, Atkinson JC. Adenoviral-mediated Gene Transfer to Mouse Salivary Glands. In submission .

Pathology Evaluation of Genetically-Engineered Mice

Charles A. Montgomery, DVM
Comparative Pathology Laboratory, Center for Comparative Medicine, 1 Baylor Plaza, Baylor College of Medicine, Houston, Tx. 77030, Phone: 713-798-6591, Fax: 713-798-8395, e-mail: cmontgom@bcm.tmc.edu and GenoPath Technologies, Inc., 12800 Melville Dr., Unit 105-A, The Point at Walden, Montgomery, Texas 77356, Phone/fax: 409.448.4880,
e-mail: cmontgom@ix.netcom.com, Web address: www.genopath.com

Genetically-engineered rodents and lagomorphs are being produced at a rapid rate internationally, and provide a unique resource for medical research in academia, industry, and government. These valuable animal models often have abnormal organ function and overt clinical disease. Anatomic and clinical pathology support in these studies provide qualitative and quantitative data that can be used to define phenotypic expression in these models. The relationship between the pathologist providing the service and the research investigator who makes the mouse should be an intellectual collaboration. This presentation discribes a multitiered phenotypic expression plan that has been used successfully for nine years. It includes frequent discussions with the investigator to obtain information on the investigator's goals, the gene, or gene family in question, the genetic background of the animal, and potential target organs. The pathology protocol for a given investigator is specifically tailored to fit his/her research needs and the GEM. The pathology evaluation includes hematology, clinical chemistry, and urinalysis, a complete necropsy with gross photography of observed lesions and organ weights, collection of tissues in multiple fixatives if necessary, and tissues for cryotomy. 50-55 tissues are prepared for histopathology and stained with H&E, then read and interpreted by a pathologist. The initial tissue screen may require polarized light, phase contrast, darkfield, or FA microscopy. Salient microscopic lesions are photographed with a digital camera and preliminary findings including photomicrographs provided to the investigator by e-mail. A final report is then completed. The second phase of investigation may include one or more of the following: special clinical chemistry, bone marrow evaluation, immunohistochemistry, in situ hybridization, electron microscopy, confocal microscopy, morphometric analysis, cell proliferation assays, or immunoprofiling by flow cytometry and/or immunohistochemistry. Special pathology protocols requiring unique instrumentation or tissue preparation can be provided for the musculoskeletal, respiratory, cardiovascular, urogenital, gastrointestinal, and neurological systems. Other pathology services for embryonic lethals, neonatal deaths, and for application studies for defined models will be discussed. Pictoral examples of transgenic mouse models will be used to demonstrate the value of morphologic methods in defining phenotypic expression in GEM.

Soriano, P., Montgomery,C.,Geske, R., and Bradley, A.: Targeted Disruption of the c-src Proto-oncogene Leads to Osteopetrosis in Mice. Cell 64: 693-702, 1991

Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montgomery, C.A. Jr., Butel, J.S. and Bradley, A.: Mice Deficient for p53 are Developmentally Normal but Susceptible to Spontaneous Tumors. Nature 356: 215-221, 1992

Yokoyama, T., Copeland, N.G., Jenkins, N.A., Montgomery,C.A., Elder, F.F.B., and Overbeek, P.A.: Reversal of Left-Right Asymmetry: A Situs Inversus Mutation. Science 260: 679-682, 1993

Harvey, M., McArthur, MJ., Montgomery, C.A.,Jr., Butel, J.S., Bradley, A., and Donehower, L.A.: Spontaneous and Carcinogen-induced Tumorigenesis in p53- deficient Mice. Nature Genetics 5: 225-229, 1993

Wu, X., Wakamiya, M., Vaishnav, S., Geske, R., Montgomery,C.,Jr.,Jones, P., Bradley, A., and Caskey, C.T.: Hyeruricemia and Urate Nephopathy in Urate Oxidase-Deficient Mice. Proc. Natl. Acad. Sci. USA 91: 742--746, 1994

Wakamiya, M., Blackburn, M.R., Jurecic, R., McArthur, M.J., Montgomery, C.A.,Jr., Cartwright, J., Jr., Mitani, K., Vaishnav, S., Belmont, J.W., Kellems, R.E., Finegold, M.J., Geske, R.S., Bradley, A., and Caskey, C.T. : Disruption of the Adenosine Deaminase Gene Causes Hepatocellular Impairment and Perinatal Lethality in Mice. Proc. Natl. Acad. Sci. USA 92: 3673-3677, 1995

Lydon, J.P., DeMayo, F.J., Mani, S.K., Hughes, A., Montgomery, C.A., Jr., Shyamala, G., Conneely, O.M., and O'Malley, B.W. : Mice Lacking Progesterone Receptor Exhibit Pleiotropic Reproductive Abnormalities. Genes and Development, 9: 2266-2278, 1996.

EFFECTS OF GENETIC BACKGROUND ON THE PHENOTYPE OF INDUCED MUTANTS

Joel Franklin Mahler, DVM
National Institute of Environmental Health Sciences
PO Box 12233, Mail Drop B3-06, Research Triangle Park, NC 27709
Phone: 919-541-0770; Fax: 919-541-7666; E-mail: mahler@niehs.nih.gov

The majority of human disorders are the result of multiple genetic and environmental factors. Even single gene diseases may have variable expression and penetrance, presumably due in part to modifier genes. The mouse has long served as the model of choice for human genetic disease, beginning with the identification and characterization of numerous spontaneous mutations, and more recently with mouse modeling technology that has allowed for targeted genetic manipulations in order to create designer stocks that are homologous to genetically based human diseases (1). Similar to the human situation, phenotypic variability is also seen in mice when particular spontaneous or induced mutations of interest have been placed on different strain backgrounds. Modulating genetic effects in mice with single gene manipulations therefore can serve as an excellent model for the complex molecular interactions known to occur in most human diseases (2).
Several recent examples illustrate the importance of strain-specific modifier genes on the phenotype of induced mutations in mice. Tumor models provide a relatively simple example of a gene background effect in the fact that, although unusual and unique neoplasms may arise that are specific for the genetic alteration, the overall tumor spectra in genetically altered mice tend to follow that of the strain background. Knowledge of both the mutation- and strain-defined tumor profiles for individual lines is important, particularly for those genetically engineered mice that are being proposed as short-term models for carcinogenesis testing, such as the p53-deficient and Tg.AC transgenic lines (3). Usually the modifying cancer resistance and susceptibility loci of the genetic background are undefined. Increasingly, however, modifier loci are being identified (4), one of the first being a gene that controlled the phenotypic expression of the multiple intestinal neoplasia (Min) mutation, identified as Mom (modifier of Min)-1 (5).
Strain background effects presumably due to modifier genes have also produced variability in phenotypes other than neoplasia. One noteworthy example is that found with epidermal growth factor receptor (EGFR) knockouts in which the time of lethality for one mutation ranged from peri-implantation to post-partum depending on the strain background (6). Post-natal disease severity is another phenotype that may be modified by genetic background. Examples such as the Ctfr knockout mouse model of cystic fibrosis (7) and several models of atherosclerosis (8) demonstrate how modulation of the phenotype of induced mouse mutations by modifier genes can more closely mimic the polygenic or multifactorial nature of important human disease and allow for models with which to understand the genetic basis for disease resistance and susceptibility.

1. Searle AG et al. Mouse homologues of human genetic disease. J. Med. Genet. 31:1-19, 1994.
2. Erickson RP. Mouse models of human genetic disease: Which mouse is more like a man? BioEssays 18:993-998, 1996.
3. Mahler JF et al. Spontaneous and chemically induced proliferative lesions in Tg.AC transgenic and p53-heterozygous mice. Toxicol. Pathol. 26:501-511, 1998.
4. Balmain A and Nagase H. Cancer resistance genes in mice: models for the study of tumor modifiers. Trends Genet. 14:139-144, 1998.
5. Dietrich WF et al. Genetic identifcation of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell 75:631-639, 1993.
6. Threadgill DW et al. Targeted disruption of mouse EGF receptor: Effect of genetic background on mutant phenotype. Science 269:230-233, 1995.
7. Rozmahel R et al. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nature Genet. 12:280-287, 1996.
8. Paigen B. Genetics of responsiveness to high fat and high cholesterol diets in the mouse. Am. J. Clin. Nut. 62:458S-462S, 1995.

Pathology of GEM Mouse Lines used in New Short-Term Carcinogenesis Bioassays

Peter C. Mann, DVM
Experimental Pathology Laboratories, Inc
PO Box 12766, Research Triangle Park, NC 27709
Phone: 919-932-5330, Fax: 919-544-7289, Mannepl@aol.com

Several lines of genetically engineered mice are currently under investigation for use in short term carcinogenesis bioassays, as alternatives, adjuncts or replacements for a two year mouse bioassay. The lines include the Tg.AC transgenic mouse, the p53-heterzygous mouse and the rasH2 transgenic mouse. All three of these lines are currently undergoing a series of validation studies either in the US, Europe, or Japan. Although there are few lesions present in these mice at the end of a 6 month carcinogenesis bioassay, a number of proliferative lesions will be described which are characteristic of one or more of the three GEM lines. In addition, treatment-related lesions from current validation studies will described. Finally, lesions resulting from unusual routes of administration will be described.

Bucher JR, Update on national toxicology program (NTP) assays with genetically altered or "Transgenic" mice. Environ Health Perspect 106:619-21, 1998

Mahler JF, Flagler ND, Malarkey DE, Mann PC, Haseman JK and Eastin W (1998). Spontaneous and Chemically Induced Proliferative Lesions Lesions in Tg.AC Transgenic and p53-Heterozygous Mice. Toxicol Pathol. 26:501-511.

Mitsumori K, Koizumi H, Nomura T and Yamamoto S (1998). Pathologic Features of Spontaneous and Induced Tumors in Transgenic Mice carrying a Human Prototype c-Ha-ras Gene Used for Six Month Carcinogenicity Studies. Toxicol. Pathol. 26:520-531

Lekidelu Taddesse-Heath, M.D.
Laboratory of Immunopathology, NIAID, NIH, Bethesda, MD 20892
LTADDESSE@atlas.niaid.nih.gov, (301)496-1150, Fax (301)402-0077

A variety of spontaneous and induced lymphoproliferative lesions and lymphomas have been observed in mice. A survey of the more commonly observed lymphomas is presented along with methodologies for characterization and
classification, including morphology, immunohistochemistry, flow cytometry for cell surface antigens and molecular analysis of immunoglobulin and T- cell receptor rearrangements. The spectrum of lesions observed in these mice
include lymphomas and leukemias of B-cell, T-cell, myeloid and erythroid derivation. Lymphomas in genetically engineered mice are often of the lymphoblastic type, although marginal zone lymphomas have been recently
described. Lymphomas in conventional mice are often of B cell origin from spleen, mesenteric nodes or Peyer's patches. Other types of lymphomas, however, have been found in both induced and natural mutants as well as conventional mice. Examples of each of these will be discussed and presented. The origin and classification of lymphomas and leukemias in mice often depends on histogenetic studies using mice of various ages and molecular and morphological studies mentioned previously. Various terminologies exist for the classification of lymphomas. Human and mouse
nomenclature systems including the Kiel, REAL, and STP classifications are presented with diagnostic criteria and correlation of the various lymphomas.

1. Pattengale, PK, et al. (1994). Experimental models of lymphoproliferative disease. Amer J Pathol. 113:237-265.

2. Fredrickson TN, et al. (1995). Classification of mouse lymphomas. Curr Topics Microbiol Immunol 194:109-116.

3. Frith CH, Ward, JM et al. (1997). Proliferative and nonproliferative lesions of the hematopoetic system in mice. In: Guides for Toxicology Pathology. STP/ARP/AFIP, Washington, DC.

4. Frith CH, Ward JM, Frederickson T and Harleman JH (1996): Neoplastic Lesions of the Hematopoietic System. In: Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP and Ward JM, editors. Pathobiology of the Aging Mouse. Washington, DC: ILSI Press, 219-235.

5. Harris NL, Jaffe ES, et al. (1994). A Revised European American classification of lymphoid neoplasms. A proposal from the International Lymphoma Study Group. Blood: 84:1361-1392.

6. Lennert K, Feller AC, (1992). Histopathology of non-Hodgkin's lymphomas (Based on the updated Kiel Classification). Berlin, Germany, Springer-Verlag.

7. Fredrickson TN, et al. Splenic Marginal Zone Lymphomas in Mice. Amer J Pathol. In Press (1999).

8. Ward, JM et al. Splenic Marginal Zone B-cell and Thymic lymphomas in p53 deficient mice. Laboratory Investigation. In Press (1999).

Unique Proliferative Lesions in Genetically Engineered Mice

Miriam R. Anver, D.V.M., Ph.D.
Head, Pathology/Histotechnology Laboratory
SAIC/NCI-FCRDC, P.O. Box B, Frederick, MD 21702-1201
m_anver@mail.ncifcrf.gov, 301-846-1281, FAX 301-846-6848

Diagnostic criteria for rodent lesions have been developed over many years and published in numerous manuscripts and text books. These publication illustrate a lack of uniformity and inconsistent terminology for neoplasia diagnoses in certain organ systems.

Classification of proliferative lesions of rodents as neoplastic or nonneoplastic can have considerable importance in safety testing and government regulatory decisions. The Society of Toxicologic Pathologists (STP) has established and published diagnostic criteria for proliferative lesions in all major organ systems of rats (Guides for Toxicologic
Pathology) to guide pathologists in lesion diagnosis; similar guides are in progress for the mouse. In general, proliferative lesions can be recognized as neoplastic if there is disruption or invasion of normal tissues by proliferating cells; perturbation of cellular architecture (loss of polarity, piling up of cells); cellular atypia (karyomegaly, cytomegaly increased nuclear cytoplasmic ratio, cytoplasmic basophilia, increased mitoses, atypical mitotic figures) and presence of metastasis for malignant tumors. Specific classification of neoplasms in organ systems have been developed by panels of pathologists and then submitted for review by the entire STP membership before being published. Certain types of genetically-engineered mice, however, develop proliferative lesions that are unique as to site, cellular morphology, and biological behavior, presenting dilemmas in diagnosis of neoplasia versus hyperplasia. A number of examples will be used to illustrate such diagnostic difficulty including proliferative gastric epithelial lesions, atypical large bowel hyperplasia with submucosal crypt cells, proliferating skeletal muscle cells in ectopic sites and proliferating epithelium/heterotopic bone/cartilage forming masses on foot pads.

Green, JG, et.al. (Oct 1995) Ectopic bone formation and chondrodysplasia in transgenic mice carrying the rat C3(1) Tag fusion gene 44th Annual Meeting of the American Society of Human Genetics.

Standardized System of Nomenclature and Diagnostic Criteria Guides for Proliferative Lesions in the Rat, STP/ARP, AFIP, Washington D.C. Society of Toxicologic Pathologists web site: http://www.toxpath.org

Takayama H., et.al. (1997) Diverse tumorigenesis associated with aberrant development in mice overexpressing hetatocyte growth factor/scatter factor PNAS 94:701-706

Takagi H, et.al. (1992) Hypertrophic gastropathy resembling Menetrier's disease in transgenic mice overexpressing transforming growth factor alpha in the stomach J Clin Invest 90:1161-1167

Colin L. Stewart D.Phil
Director, Laboratory of Cancer and Developmental Biology
ABL-Basic Research Program, NCI-FCRDC
P.O.Box B, Frederick MD 21702-1201
Tel: 301-846-1755, Fax: 301-846-7117, stewartc@ncifcrf.gov

The introduction of genetically altered embryonic stem (ES) cells into mouse preimplantation embryos is the standard route to deriving new lines of mice carrying mutated genes in their germ line. Some of these novel mutations result in either preimplantation or early postimplantation lethality, complicating the analysis of the mutation. There are however additional procedures to deriving these embryos that substantially help in the analysis of these early embryonic lethals and in understanding the mutated gene's role in producing the lethal phenotype. Thus homozygous null embryos from female homozygotes that die before reaching sexual maturity, maternal effect mutants, or those in which female reproduction is affected can be analyzed by transplantation of the ovaries from the mutant to wild-type recipients. By using this procedure failures in ovarian function or in maternal reproductive physiology can be restored allowing embryo development to proceed. Information on early embryonic lethals can also be garnered by altering the genotype of the host embryo or making the ES cells homozygous for the mutation. Tetraploid blastomeres in the presence of diploid cells (other embryos, ES cells) preferentially form the extraembryonic membranes (trophoblast and placenta, extraembryonic endoderm). Using such embryos has revealed that particular mutations affect placental or extra-embryonic membrane function in development. Similarly, the introduction of ES clones homozygous for a particular mutation has permitted the derivation of embryos comprised entirely from the ES cells without having to derive and identify the homozygotes by conventional breeding. Similarly the production of partial chimeras, in which the ES cells make a limited contribution to the developing embryo has been useful in identifying which tissue is principally affected by the mutant gene. Lastly, in vitro culture and manipulation of early postimplantation embryos or the establishment of cell lines from chimeras should not be ignored as a potential means to analyzing mutant phenotypes.

Dufort D, et al. The transcription factor HNF3beta is required in visceral endoderm for normal primitive streak morphogenesis. Development. 1998 Aug;125(16):3015-25.

Everett CA, et al. The influence of ploidy on the distribution of cells in chimaeric mouse blastocysts. Zygote. 1996 Feb;4(1):59-66.

Guillemot F, et al. Essential role of Mash-2 in extraembryonic development. Nature. 1994 Sep 22;371(6495):333-6.

Rivera-Perez JA, et al. Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. Development. 1995 Sep;121(9):3005-12.

Rossant J, et al. Chimeras and mosaics in mouse mutant analysis. Trends Genet. 1998 Sep;14(9):358-63

Sztein J, et al. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol Reprod. 1998 Apr;58(4):1071-4.

Yamamoto H, et al. Defective trophoblast function in mice with a targeted mutation of Ets2. Genes Dev. 1998 May 1;12(9):1315-2

Wang ZQ, et al. Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mech Dev. 1997 Mar;62(2):137-45

Professor Matthew H Kaufman, PhD, DSc., FRCP Edin.
Department of Biomedical Sciences (Anatomy), University Medical School, Teviot Place, Edinburgh EH8 9AG, UK
Telephone: 0131-650-3113 (direct), Fax: 0131-650-6545, e-mail: M.Kaufman@ed.ac.uk
http://genex.hgu.mrc.ac.uk/

The mouse is the most commonly employed experimental animal, and much information may be obtained from the analysis of its
embryonic and fetal stages. Accordingly, most effort is presently concentrated in attempting to understand the genetic factors that influence its normal development, through analysing the effect of an abnormal genome on its phenotype. One of the most informative approaches for undertaking such an analysis is through the examination of appropriately fixed material which has been embedded in either paraffin wax or plastic, and then serially sectioned in one of several 'conventional' planes - usually such material is sectioned in either the transverse, sagittal or coronal plane. The most commonly used fixatives are Bouin's solution, 10% formalin and 4% paraformaldehyde, but specific stains may need other fixatives. In order to maximally facilitate the interpretation of the sectioned material, the orientation of the embryo in the paraffin or plastic 'block' is of critical importance, and every effort should be made to section the embryo both symmetrically and in one or other of the planes indicated above. It is particularly important to follow exact guidelines during the preparation of this material, as the duration of its various stages is largely dependent on the stage of embryonic/fetal development studied. Most researchers employ the staging system proposed by Theiler. In the interpretation of the histological sections, it is useful, though not absolutely essential, to have a sound knowledge of the anatomical tissues and features likely to be present at the stage being studied, and text-based databases are now available for this purpose. It is also essential to have access to a standard reference manual in which all of the key features on the individual sections are clearly labelled. If a specific system is studied, then a sound knowledge of its embryological development is also of critical importance. The availability in the near future of 3-dimensional reconstructions of all stages of mouse development, prepared from serially sectioned material, will provide an additional important teaching aid for those that require to work with morphologically normal material with which they are not familiar, as each of its component tissues will be colour-coded. Additionally, to facilitate the recognition of specific tissues, once the developmental stage is established, the 'reference' or 'standard' reconstructed embryo may be 'resectioned' in any orientation, so that computer-generated sections may be obtained which match the viewer's own sections. Using this resource, it will soon be possible for researchers to insert (or 'paint') domains of gene expression onto these 'standard' embryos, though it has to be appreciated that domains of gene expression may not always adhere strictly to conventional anatomical domains. By using appropriate bioinformatics approaches, it should be possible to search such gene-expression databases, both spatially and textually, but for them to be of maximum value, they will have to be easy to use, comprehensive and accessible over the internet.

Bard, JBL., Baldock, R.A., & Davidson, D.R. (1998). Elucidating the genetic networks of development: a bioinformatics approach. Genome Research 8, 859-863.

Bard, J.B.L., Kaufman, M.H., Dubreuil, C., Brune, R.M., Burger, A., Baldock, R.A. & Davidson, D.R. (1998). An internet-accessible database of mouse developmental anatomy based on a systematic nomenclature. Mechanisms of Development 74, 111-120.

Davidson, D., Bard, J., Brune, R., Burger, A., Dubreuil, C., Hill, W., Kaufman, M., Quinn, J., Stark, M. & Baldock, R. (1997). The mouse atlas and graphical gene-expression database. Semin. Cell Devel. Biol. 8, 509-517.

Davidson, D., Baldock, R., Bard, J., Kaufman, M., Richardson, J.E., Eppig, J.T. & Ringwald, M. (1998). Gene expression databases. In: In Situ Hybridization: A Practical Approach. Second Edition (ed. D.G. Wilkinson), Oxford: Oxford University Press. pp. 189-214.

Kaufman, M.H. (1994). The Atlas of Mouse Development. Second Printing, with Index. London: Academic Press. 525pp.

Kaufman, M.H. & Bard, J.B.L. (1999). The Anatomical Basis of Mouse Development. San Diego: Academic Press. (in press)

Kaufman, M.H., Brune, R.M., Baldock, R.A., Bard, J.B.L. & Davidson, D. (1997) Computer-aided 3-D reconstruction of serially sectioned mouse embryos: its use in integrating anatomical organization. International Journal of Developmental Biology 41, 223-233.

Kaufman, M.H., Brune, R.M., Davidson, D.R. & Baldock, R.A. (1998). Computer-generated three-dimensional reconstructions of serially sectioned mouse embryos. J. Anatomy 193, 323-336.

Ringwald, M., Baldock, R., Bard, J., Kaufman, M., Eppig, J.T., Richardson, J.E., Nadeau, J.H. & Davidson D. (1994). A database for mouse development. Science 265, 2033-2034.

Jerrold M. Ward, D.V.M., Ph.D. and Deborah E. Devor-Henneman, B.S.
Veterinary and Tumor Pathology Section, Animal Sciences Branch, Office of Laboratory Animal Resources, Division of Basic Sciences, National Cancer Institute, Frederick, MD 21702-1201
301-846-1239, fax 301-846-6389
www.ncifcrf.gov/vetpath, ward@mail.ncifcrf.gov, ddh@mail.ncifcrf.gov

The mouse placenta is an important, but often neglected, organ derived from extra-embryonic and maternal tissues. It is the only tissue providing for embryonic nutrition and development. Numerous important genes are expressed in various anatomical regions of the mouse placenta and play critical roles in placental and embryonic growth and maturation. Early placentation takes place around the ectoplacental cone at 8-9 days post-coitum (dpc) and reaches maturity by 12dpc. In specific gene knockout studies, embryonic death early in gestation (<12 dpc) with defects in cardiovascular, hematopoietic and/or neurological development have been well documented. Less well documented, however, is the occurrence of placental atrophy and dysplasia in conjunction with embryonic death. Placental origin of embryonic death may be difficult to determine. Anatomical components of the placenta include membranes (yolk sac, amnion, allantois, chorion), angiogenic mesenchyme, embryonal and maternal blood cells, embryonal and maternal blood vessels, several types of trophoblasts and support tissues. Gene expression, as studied by in situ hybridization and immunohistochemistry, often occurs in trophoblasts. Loss of specific gene expression in knockout mice may produce abnormal placentation and embryonic death by day 12. More than 50 gene knockout mice have been reported to have various types of placental defects. Placental dysplasia can be limited to yolk sac, membrane fusion, angiogenesis, trophoblasts and decidua. Differentiation of primary from secondary placental atrophy can be difficult. It requires consideration of tissues that normally express the specific gene knocked out, early lesions in embryo or placenta and other factors. Rescue of the placenta by specific techniques allows for development of the knockout embryo and study of gene function in the knockout.

Copp AJ. Death before birth: clues from gene knockouts and mutations. TIG 11: 87-93, 1995.

Cross JC, Werb Z and Fisher SJ. Implantation and the Placenta: Key Pieces of the Development Puzzle. Science 266: 1508-18, 1994.

Gnarra, J.R., Ward, J.M., Porter, F.D., Wagner, J.R., Devor, D.E., Grinberg, A., Emmert-Buck, M.R.,
Westphal, H., Klausner, R.D., Linehan, W.M. Defective placental vasculogenesis causes embryonic
lethality in VHL deficient mice. Proc. Natl. Acad. Sci. USA 94:9102-9107, 1997.

Kaufman MH. The Atlas of Mouse Development. London: Academic Press, 469-489, 1992.

Steingrimsson E, Tessarollo L, Reid SW, Jenkins NA, Copeland NG. The bHLH-Zip transcription
factor Tfeb is essential for placental vascularization. Development 125:4607-4616, 1998.

Theiler K. The House Mouse. New York: Springer-Verlag, 178pp., 1989.

Yamamoto H, Flannery ML, Kupriyanov S, Pearce J, McKercher SR, Henkel GW, Maki RA, Werb Z, Oshima RG
Defective trophoblast function in mice with a targeted mutation of Ets2. Genes Dev 12:1315-26, 1998.

The skeletal system develops from three different lineages, the paraxial (somitic) mesoderm, the lateral plate mesoderm, and the cranial neural crest which give rise to the vertebral column, the limbs, and the skull, respectively. Consequently, some of the secreted signals and regulatory cascades that determine the pattern of skeletal elements derived from each of these sources are shared, while others are distinct. Genetic changes that affect skeletal pattern may impact on only one, or any combination of these three lineages. Different parts of the skeleton also differ in the manner by which the final bony skeleton is formed, either via an intermediate cartilage model, or by direct ossification from mesenchyme. A combination of molecular genetic and classical embryological approaches, particularly in mouse and chick, have generated a fairly detailed picture of the major tissue interactions and molecular signals that regulate skeletal patterning in each lineage, as well as those that regulate later processes of chondrogenic and osteogenic differentiation and bone growth. Some of the signaling cascades and developmental control genes involved in these various aspects of skeletogenesis also regulate steps in the formation of other organ systems. Skeletal analysis may reveal developmental regulatory components that are perturbed in genetically engineered mice in cases where other aspects of development are also affected. Skeletal patterning is closely coupled with growth; regulation of proliferation comes into play very early when primary skeletal precursors (anlage) are just beginning to form and continues quite late into the postnatal period, beneath the superficial periosteum and also in the epiphyseal growth plates of long bones. Bone growth is accompanied by considerable remodeling through selective destruction by osteoclasts. Thus, the ultimate morphology of the adult skeleton is also in part determined by the balance between these processes.
The normal development and pathologic analysis of phenotypes affecting both early and late events in the formation of the skeleton will be discussed, in the context of a review of genetically engineered mouse mutants known to affect skeletogenesis. Discerning and evaluating abnormalities that involve regulatory components will be emphasized. Since the skeleton can be easily separated from the intact thoracic and abdominal viscera, analysis of skeletal morphology can be carried out together with a simultaneous evaluation of organogenesis in the same animals and may provide important mechanistic clues into the pathogenesis of complex phenotypes that affect multiple organ systems.

Selected recent reviews:

Christ, B. et al. (1998) Segmentation of the vertebrate body. Anat. Embryol. 197: 1-8.

Erlebacher, A. et al. (1995) Toward a molecular understanding of skeletal development. Cell 80: 371-378.

Francis-West, P. et al. (1998) Signalling interactions during (cranio)facial development. Mech. Dev. 75: 3-28.

Innis, J.W. and Mortlock, D.P. (1998) Limb development: molecular dysmorphology is at hand. Clin. Genet. 53: 337-348.

Karsenty, G. (1998) Genetics of Skeletogenesis. Dev. Genet. 22: 301-313.

Wallis, G.A. (1996) Bone growth: coordinating chondrocyte differentiation. Curr. Biol. 6: 1577-1580.

D.M. Jacobowitz, A. T. Kallarakal, C. Tohda, F. Lau and L.C. Abbott

Laboratory of Clinical Science, Building 10, Rm. 3D-48; Bethesda, MD 20892
E-mail: dwj@helix.nih.gov

The study of normal and pathological development of the brain is vastly enhanced by the use of chemical markers and stains. The use of antisera and histochemical stains allows researchers to accurately identify major anatomical structures of the developing brain not easily delineated by conventional Nissl and myelin stains. A chemoarchitectonic atlas of the developing mouse brain has been assembled (Jacobowitz, D.M. and Abbott, L.C., "Chemoarchitectonic Atlas of the Developing Mouse Brain," CRC Press, Boca Raton, Florida, 1998) in order to combine chemical neuroanatomy (chemoarchitectonics) with cytoarchitectonic markers. This yields a powerful source of information providing state-of-the-art brain cartography. A variety of chemical markers were used (tyrosine hydroxylase, serotonin, calretinin, calbindin, acetylcholinesterase) at gestational ages E11/l12, E13/14, E15/16, E17/8 and PO (newborn). This provides an enormous amount of information concerning the location and first appearance of specific cell bodies and/or intensely staining neuronal processes.

The development of the Laser Capture Microscope (Science 274:998, 1996) has sparked the formulation of a new research initiative. In an effort to identify genes in the substantia nigra which are relevant to neurodegeneration in Parkinson's disease, we undertook a study utilizing laser capture microdissection of mouse embryo (E12) substantia nigra neuroepithelial cells. RT-PCR and differential display were performed with the total RNA isolated from the laser-captured tissue. Differentially expressed bands were identified by sequencing. Comparison of the embryonic substantia nigra neuroepithelium with a control region without dopamine cells (tectum) revealed a differential expression of a CAG repeat cDNA in addition to other known proteins. The use of laser capture microdissection opens up a new horizon in gene discovery in the developing mouse brain and peripheral tissue.

Gene targeting of neurotrophins and their receptors: pleiotropic developmental defects

Lino Tessarollo, Ph.D.
Neural Development Group, Cancer and Developmental Biology Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Building 539, Room 205, PO Box B, Frederick, MD 21702-1201
Phone: 301-846-1202, Fax: 301-846-6666, E-mail tessarol@ncifcrf.gov , http://nmrweb.ncifcrf.gov/abl/mgl/tessarollo.html

Neurotrophins are soluble growth factors known mainly for their roles in regulating the development of the mammalian nervous system. Two types of receptors mediate the actions of these polypeptides: the Trk family of tyrosine kinase receptors and the so-called p75 low-affinity NGF receptor. Gene targeting experiments in the mouse have uncovered some of their functions in promoting survival and developmental maturation of certain types of neurons of the peripheral and central nervous system confirming the critical role of neurotrophins and their receptors in neural development. However, these families of genes are also widely expressed in a variety of nonneuronal systems throughout development, including the cardiovascular, endocrine, reproductive, and immune systems. The severity of deficiencies in mice lacking specific neurotrophin functions suggests that activation of these signaling pathways is required in a variety of organs during ontogenesis. For example, expression of neurotrophin-3 (NT-3) and its trkC receptor has been reported in the neural crest and the cardiovascular system. Mice lacking NT-3 or trkC develop numerous cardiac malformations, including tetralogy of Fallot, ventricular septal defects, and atrial septal defects that resemble the most common congenital pediatric cardiac malformations of humans. This phenotype is consistent with abnormalities in the survival and/or migration of the cardiac neural crest early in embryogenesis. The neural crest cells contribute to the mesenchymal components of many organs in development. Thus, it would not be surprising if other defects in nonneuronal neural crest-derived structures would be identified upon further analysis of these mutant mice. A comprehensive analysis of these mouse models is being conducted in order to identify the role of NT-3 and trkC outside the nervous system and in differentiating the complex functions of neural crest cells.

Tessarollo, L., Tsoulfas, P., Martin-Zanca, D., Gilbert, D.J., Jenkins, N.A., Copeland, N.G. and Parada, L.F. 1993. trkC, a receptor for neurotrophin-3, is widely expressed in the developing nervous system and in non-neuronal tissues. Development 118, 463-475.

Tessarollo, L., Vogel, K.S., Palko, M.E., Reid, S.W., and Parada, L.F. 1994. Targeted mutation in the neurotrophin-3 gene results in loss of muscle sensory neurons. Proc. Natl. Acad. Sci. 91, 11844-11848.

Donovan, M.J., Hahn, R., Tessarollo, L., and Hempstead, B.L. 1996. Neurotrophin-3 is required for normal mammalian cardiac development: identification of an essential nonneuronal neurotrophin function. Nature Genetics 14: 210-213

Liebl, D.J., Tessarollo, L., Palko, M.E. and Parada L.F. 1997. Absence of Sensory Neurons before Target Innervation in Brain-Derived Neurotrophic Factor, Neurotrophin-3 , and TrkC-Deficient Embryonic Mice. J. Neurosci. 17: 9113- 9121.

Tessarollo, L., Tsoulfas, P., Donovan, M.J., Palko, M.E. , Blair-Flynn, J., Hempstead, B.L. and Parada, L.F. 1997. Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand Neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis. Proc Natl Acad Sci. 94: 14776-14781.

Tessarollo, L., and Hempstead, B.L. 1998. Regulation of cardiac development by receptor tyrosine kinases. Trends in Card Med 8: 32-37.

Kucera, J., Fan, G., Walro, J., Copray, S., Tessarollo, L., and Jaenisch, R. 1998. Neurotrophin-3 and trkC in muscle are non-essential for the development of mouse muscle spindles. NeuroReport 9: 905-909.

Tessarollo, L. 1998. Pleiotropic developmental functions of neurotrophins in development. Cytokine Growth Factor Rev 9: 125-137.

Palko, M.E., Coppola, V. and Tessarollo, L. Evidence for a role of truncated trkC receptor isoforms in mouse development. J. Neurosci. 19: 775-782.

Jacqueline N. Crawley, Ph.D. Chief, Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Building 10 Room 4D11, Bethesda, MD 20892 USA
301-496-7855, FAX 301-480-1164, jncrawle@codon.nih.gov

Rigorous experimental design is required for the behavioral phenotyping of a new transgenic or knockout mouse. Use of well established, quantitative behavioral tasks, appropriate Ns, correct statistical methods, consideration of background genes contributed by the breeder parents, and consideration of litter and gender issues, will yield meaningful comparisons of -/-, +/-, and +/+ genotypes.

Our laboratory has designed methods to optimize the behavioral characterization of mutant mice.1 Initial measures evaluate general health, neurological reflexes, sensory abilities, and motor functions. Specific tests include measures of home cage behaviors, body weight, body temperature, appearance of the fur and whiskers, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, Digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity.

Hypothesis testing then focuses on at least three well-validated tasks within each relevant behavioral domain. Specific tests will be described and illustrated for the domains of learning and memory, feeding, nociception, and behaviors relevant to discrete symptoms of human anxiety, depression, schizophrenia, and drug addiction. Adaptation of standard rat tests for use in mice will be emphasized.

An example of our approach will be described for the behavioral phenotyping of C/EBPd knockout mice, generated by Drs. Esta Sterneck and Peter Johnson, ABL, NCI, recently published in Proceedings of the National Academy of Sciences USA.2 C/EBP transcriptional regulators are five related basic-leucine zipper DNA binding proteins, some of which have been implicated in learning and memory in Drosophila and Aplysia. C/EBPd null mutants are normal on measures of body weight and temperature, physical appearance, home cage behaviors, neurological reflexes, open field activity, rotarod performance, light«dark exploration, acoustic and tactile startle, prepulse inhibition of startle, and the Morris water task, as compared to wildtype littermate controls. Significantly better performance was detected in C/EBPd -/- mice on the cued and contextual conditioning task, selective for conditioned freezing to context. Taken together with the normal behavioral phenotype on all other tests, this finding implicates C/EBPd specifically in fear-conditioned learning and memory.

1Crawley JN, Paylor R: A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Hormones and Behavior 31: 197-211, 1997.

2Sterneck E, Paylor R, Jackson-Lewis V, Libbey M, Przedborski S, Tessarollo L, Crawley JN, Johnson PF: Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/Enhancer Binding Protein d. Proceedings of the National Academy of Sciences USA 95: 10908-10913, 1998.
Mouse Imaging

R. N. Bryan, M.D., Ph.D. and R. S. Balaban, Ph.D.
Director, Diagnostic Radiology, Radiology Department, Clinical Center, NIH, Building 10, Room 1C660, Bethesda, MD 20892
Telephone: 301-435-5741, Fax: 301-496-9933
Email: nbryan@nih.gov

Biological systems, from simple cells to complex organisms, have an intrinsic spatially distributed nature. That is, portions of an organism vary from one place to another in terms of morphology, physiology, chemistry and function. To study an organism one must make spatially distributed measurements in order to understand the nature of local tissues and their relationships to each other. Imaging is nothing more than a means of making and displaying spatially coherent measurements and is therefore a key resource for studying biological systems.
A major limitation to studying many biological questions with current imaging techniques is their limited applicability to living organisms, especially larger organisms. Key understanding of biological systems is now being derived from molecular biological techniques that are often related to small animal models, particularly genetically engineered mice. Most in vivo imaging devices have been optimized for human studies and have inadequate spatial resolution for small animals and their pathology. However, most imaging techniques can be scaled down to yield very high resolution and signal sensitive in vivo images of mouse sized samples. Furthermore, there are some imaging techniques that could provide valuable information in small animal models, but are not applicable to human subjects. The fundamental goal is to develop imaging techniques which can help evaluate mouse 'phenotypes' for complete genotype/expression description.

Currently there are four main imaging techniques available for mouse work: MRI, PET/SPECT, X ray-CT and Ultrasound.
MRI:
MRI provides the state of the art in structural imaging of soft tissues as well as numerous physiological parameters, in vivo. Currently MRI studies can be routinely performed on mice collecting most of the data available in humans. The resolution of these images collected at 4.7T or higher is well under a millimeter (100 to 300 microns) in plane with 1 millimeter slices. The only complication is when the use of contrast agents is required for observing vascular volume or the blood-tissue barriers when a sterile venous line is required. Some biochemical information is available form MR spectroscopy but with much lower spatial resolution
PET/SPECT
Positron emission tomography, single photon emission computed tomography and projection imaging of radiotracers administered to small animals provide a flexible set of procedures for evaluating organ function in vivo (or ex vivo) in normal animals, animals modeling human diseases and in genetically altered animals. Although these methods were developed for human use, many of the associated imaging procedures have been, or can be, adapted to small animals. For example, gated equilibrium blood pool imaging of the heart, a common procedure for visualizing and quantifying cardiac function in man, is being performed in mice genetically altered to exhibit hypertrophic cardiomyopathy. This procedure allows, in principle, the functional consequences of HCM to be quantified over time in the same animal as the condition develops. A PET imaging study is now also underway in which liver uptake and excretion of fluoro-deoxyglucose are being monitored in mice genetically altered such that glucose metabolism in the liver is abnormal. A therapy to reverse this genetic condition in vivo will be evaluated in these same animals once baselines are established.
However, SPECT and planar imaging systems have not yet been optimized for small animal imaging and studies carried out with human scanners are limited in both the spatial and temporal domains. A further difficulty common to all of these tracer imaging studies is the need in many cases to administer the tracer directly into the vascular system by vein or artery.

CT-X ray
X-ray CT perhaps offers the best opportunity for high throughput, high resolution morphological imaging, especially on non-vital samples. Commercial instruments are available; however, most of the commercial instruments have been designed for industrial samples and, as a result, are optimized for high density materials such as bone. A major DOE project for high throughput rodent CT imaging is in place at Oakridge where a one-of-a-kind system is capable of scanning up to one animal/minute. High throughput, non-vital mouse CT imaging would be straightforward; vital imaging would be considerably more complicated, but perhaps less so than for MR imaging as a whole animal CT could possibly be performed in 1 to 5 seconds. The images could have a spatial resolution of approximately 50 microns with excellent bone contrast. Soft tissue contrast would be significantly less, especially with IV contrast enhancement. However, major organ morphometric analysis should be feasible.

ULTRASOUND
Ultrasound provides a rapid and cheap method of evaluating soft tissue structures in the body as well as some physiological parameters, most notable blood velocities. Mouse ultrasound has already been developed to the degree of being used for evaluating cardiovascular performance as well as simple structural measurements. Again using a high frequency probes (>12 MHz) and very high scan rates (30 frames/sec) much of the information available in human ultrasound can be obtained in the mouse including measurements on the fetus. The fetal measurements are extremely interesting since many genetic manipulations never make it to birth alive. New power spectrum and 3D modes will not only increase the information density of the images, but also allow faster screening imaging and image analysis. The device is also portable and can be kept with the mice in a clean facility. This is a very significant point with regard to screening large populations of mice.

New Technology:
Several new approaches to imaging are being developed that might be particularly interesting in the study of mice.
Optics: No other "vital" imaging modality gives higher signal to noise and better spatial resolution than optical imaging. However, due to the high absorbency of biological tissues, these approaches are not very useful in adults. Since much of the interest in the phenotype of the mouse is right at birth, or many times even before for some fatal modifications, many studies could ideally be performed on the neonatal or fetal animal. Due to the small size and high optical transmittance of the neonatal mouse, several investigators have been evaluating the use of visible light to imaging the structures within the unanesthetized fetal mouse. Using simple video microscopy methods, the NHLBI investigators have observed the vasculature of the brain and body as well as liver, kidney volumes as well as the dynamics of the neonatal mouse heart using the intrinsic optical contrast of the body (blood and myoglobin,

EPR Imaging: Electron paramagnetic resonance imaging is potentially of great value for evaluating tissue oxygenation and free radical concentration. However the technique is intrinsically limited by the low natural concentration of EPR detectable compounds and their extremely short half-lives as well as a very shallow field of view. The Radiation Biology Lab of NCI is pursuing this technology for mice.

MR Microscopy Duke University's Center for In Vivo Microscopy
Www.civm.mc.duke.edu/

Brad Smith at Duke U.
embryo.mc.duke.edu/animal/home.html

Micro CT
Oak Ridge National Laboratory
www.ic.ornl.gov/rd-groups /msd/Projecrs/Microcat_Web/main.htm

Micro PET UCLA
www.nuc.ucla.edu/html_docs/crump/resprojects/microPET.html

John P. Sundberg, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500
Fax: 207-288-6276, Jps@aretha.jax.org, http://lena.jax.org/~bas/

The skin is the largest of the intermediate sized organs. Phenotypic deviants with abnormalities of this organ system are easy to spot and therefore there are large numbers of spontaneous mouse mutations with abnormalities of the skin and adnexa that have been accumulated and studied for over a century. This is an important resource that has become increasingly acknowledged by the fact that many gene targeting studies (creations of partial or complete functional nulls or knockouts) develop phenotypes identical to the spontaneous mutations. Gene targeting technology answers the question of which gene is mutated, information not available on many of these spontaneous mutations. However, the collective knowledge on the biological and pathological aspects of the spontaneous mutations provides guidelines or specific answers on the function of the gene under investigation. For example, the null mutation of fibroblast growth factor 5 (Fgf5) turned out to not be an embryonic or neonatal lethal as the investigators anticipated but just grew hair that was abnormally long. Fgf5 mapped near the angora mouse mutation, a spontaneous mutation that have been available for decades that is characterized by prolongation of the hair growth cycle. The induced null mutation turned out, in breeding studies, to be an allele of angora.
Numerous spontaneous mutations are available in which the phenotypes are identical or very similar even though the mutant gene locus maps to different chromosomes. These are called phenotypic mimics. Defining one permits evaluation of others with the potential of working out a biochemical cascade. For example, an induced null mutation of Tgfa resulted in mice with wavy hair. This turned out to be an allele of waved-1, a spontaneous mutation that mapped near Tgfa. Another spontaneous mutation that was very similar to waved-1 was called waved-2. This mutation mapped near the receptor for Tgfa, Egfr. Waved-2 was subsequently determined to be a mutation of the Efgr gene.
Mutations that effect the skin can be categorized generally into the following categories: 1) structural defects of hair fibers, 2) missing hair fiber types, 3) inflammatory based skin diseases, 4) noninflammatory based skin diseases, 5) proliferative skin diseases, 6) scarring skin diseases, 7) mutations affecting the hair cycle, 8) hair color (pigmentation) mutations, and 9) structural and growth defects in nails. Although these are not clear cut and separate categories, the division provides an approach to evaluating and comparing mouse mutations between mice as well as with human and domestic mammals. Examples will be provided of all these phenotypes.

1. Sundberg JP. Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools. CRC Press, Boca Raton, 1994.
2. Sundberg JP, Boggess B. Systematic approach to evaluation of mouse mutations. CRC Press, Boca Raton, FL, (in press).
3. Sundberg JP, King, LE jr. Mouse mutations with skin and hair abnormalities as animal models for dermatological research. J Invest Dermatol 106:368-376, 1996.

Techniques for the Evaluation of Neuropathology in Mice

Roderick T. Bronson, D.V.M.
USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington St.,
Boston, MA 02111
bronson_pa@hnrc.tufts.edu, Telephone: (617) 556-3206, FAX: (617) 556-3205 or (617) 556-3344

Rather than review the many nervous system lesions that have been described over the years in knockout mice, the focus of this presentation will be on the technical issues of how to study the nervous system of mice to maximize the chances of finding lesions and avoiding artifacts. The best fixation method is intracardiac perfusion with Bouin's fixative. After 1-2 week fixation which also results in demineralization of bone, the brain spinal cord and legs are sliced with nervous tissue and bone in situ. Histological sections are stained with hematoxylin and eosin, which particularly stain hypertrophic astrocytes, dystrophic axons and acutely necrotic neurons. Replicate sections of tissues are stained with combined cresyl violet, for study of cell detail and luxol fast blue for study
of myelination. Embryonic tissues are usually serially sectioned either in coronal or saggital sections and stained with luxol fast blue. Some immunohistological stains such as glial fibrillary protein are useful. The pros and cons of stains for apoptosis will be discussed. Examples of malformations and degenerative lesions will be shown.

Bronson RT, Sweet HO, Spencer CA, Davisson MT. 1992. Genetic and age-related models of neurodegeneration in mice: dystrophic axons. J Neurogenet 8:71-83.

Bronson RT, Lipman R, Harrison D. 1993. Age-related gliosis in the white matter of mice. Brain Res 609:124-128.

Bronson RT, Donahue LR, Johnson KR, Tanner A, Lane PW, Faust JR 1998. Neuronal ceroid lipofuscinosis (nclf), a new disorder of the mouse linked to chromosome 9. Am J Med Genet 77:289-297.

Cook SA, Bronson RT, Donahue LR, Ben-Arie N, Davisson MT. 1997. Cerebellar deficient folia (cdf): a new mutation on mouse Chromosome 6. Mamm Genome 8:108-112.

Donahue LR, Cook SA, Johnson KR, Bronson RT, Davisson MT. 1996. Megencephaly: A new mouse mutation on chromosome 6 that causes hypertrophy of the brain. Mamm. Genome 7:871-876.

Franklin KBJ, Paxinos G. 1997. The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York.

Harris BS, Franz T, Ullrich S, Cook S, Bronson RT, Davisson MT. 1997. Forebrain overgrowth (fog): a new mutation in the mouse affecting neural tube development. Teratology 55:231-240

Kaufman MH, 1992. The Atlas of Mouse Develpment. Academic Press, London.

Sah VP, Attardi LD, Mulligan GJ, Williams BO, Bronson RT, Jacks J. 1995. A subset of p53 deficient embryos exhibit exencephaly. Nature Genet 10:175-180.

Schambra, Lauder and Silver. Atlas of the Prenatal Mouse Brain. Academic Press, New York.

Shen J, Bronson R, Chen DF, Xia W, Selkoe DJ, Tonegawa S.1997. Skeletal and CNS defects in presenilin-1 deficient mice. Cell.
89:629-639.

A Neurological Syndrome in FVB/NCr Mice

Deborah E. Devor , B.S.(ddh@mail.ncifcrf.gov), Jeffrey E. Green, M.D. and Jerrold M. Ward, D.V.M., Ph.D.

Veterinary and Tumor Pathology Section, Animal Sciences Branch, Office of Laboratory Animal Resources, Division of Basic Sciences, National Cancer Institute, Frederick, MD 21702-1201 and Transgenic Oncogenesis, Laboratory of Cell Regulation and Carcinogenesis, Division of Basic Sciences, National Cancer Institute, Bethesda, MD 20892

During the course of an experiment characterizing the effect of a particular transgene in FVB/NCr mice, we noticed a gradual change in behavior occurring in both the transgenic and wild-type control populations. By the termination of the experiment, it was determined that possibly as much as 57% of the animals eventually showed symptoms of what we have since defined as a neuroendocrinological syndrome ("Space cadet" syndrome), which may be the result of a relatively new mutation or simply an inherent condition not previously recognized in the FVB mouse. This syndrome is clinically marked by an initial period of hyper-reactivity to stimuli followed by a descent into an almost catatonic state in which there is a delayed exaggerated response. Seizures can and do occur throughout the course and are not infrequently fatal1, but are only one symptom produced by the underlying pathology. Unexpected death in apparently healthy FVB mice should be considered suspect, as should sudden aggression among never-separated littermates and abrupt changes in breeding behavior, e.g., reduced fertility, aggression towards potential mates or an established partner, and excessive infanticide postpartum. The syndrome also encompasses various gross changes internally, specifically overdistension of the gall and urinary bladders, adrenal hypertrophy, and, in end-stage animals, measurable enlargement of the brain. Variable brain lesions are seen but a new unique brain lesion will be described. Space cadet syndrome seems to have arisen in the FVB line maintained by the APA at NCI-FCRDC (FVB/NCr) within the past 5 years; however, at least 2 closed colonies initially derived from the same source as these affected animals prior to this time frame have remained symptom-free and do not exhibit the microscopic lesions seen in afflicted mice. While this syndrome may not preclude the use of the strain in transgenic studies, where it has gained favor due to the size of its pronuclei and high fertility, the fact that it does crop up in a fairly high percentage of the NCr line should be taken into account, especially in experiments involving neuroendocrinological functions2. It might also be advisable to consider rederiving the strain from non-afflicted colonies.

1Goelz, M.F., Mahler, J., Harry, J., Myers, P., Clark, J., Thigpen, J.E., and Forsythe, D.
1998. Neuropathologic findings associated with seizures in FVB mice. Laboratory Animal Science 18: 34-37
2Hsiao, K.K. Borchelt, D.R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, et al.
1995. Age-related CNS disorder and early death in transgenic FVB/N mice
overexpressing Alzheimer amyloid precursor proteins. Neuron 15: 1203-1218

Marion J.J. Gijbels, Ph.D., Menno P.J. de Winther, M.S., Ko Willems van Dijk, Ph.D., Marten H. Hofker, Ph.D.. Dept. of Human Genetics, Leiden University Medical Center , Wassenaarseweg 72, 2333 Al Leiden, The Netherlands, (MJJG, Phone: +31-71-5276410, Fax: +31-71-5276075, Email: mg@ruly46.medfac.leidenuniv.nl) and Louis M. Havekes, Prof. Dr., Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.

ApoE serves as a ligand in the receptor mediated uptake of chylomicrons and VLDL. Mutant forms of ApoE lead to an impaired clearance of remnant lipoproteins from the blood by the liver. This condition is known as Familial Dysbetalipoproteinemia (FD) and leads to hypercholesterolemia and hypertriglyceridemia. Mutations in the APOE gene can behave as a dominant or recessive trait. The recessive inheritance pattern occurs in FD patients carrying the APOE2(Arg-158®Cys) mutation. Approximately 1% of the population is homozygous for this defective APOE2 allele. The APOE3Leiden mutation (a 7-amino acid tandem repeat of residues 120-126) is an APOE mutation which behaves as a dominant trait. The main clinical features associated with FD are premature atherosclerosis and the occurrence of xanthomas. In both processes, macrophage derived apoE is thought to play an important role in the uptake and efflux of cholesterol. To investigate the role of different apoE variants in atherosclerosis and xanthoma development, we have previously generated mice transgenic for APOE2(158) and APOE3Leiden (1,2). To obtain comparable plasma cholesterol levels, these mice were bred onto an endogenous Apoe and Ldl receptor deficient background. Both transgenic and non-transgenic Apoe-/- Ldlr-/- lines have plasma cholesterol levels of approximately 35 mmol/l. We have studied the atherosclerosis in the aorta and the pathology of the lines APOE-/-LDLR-/-, APOE3LeidenAPOE-/-LDLR-/- and APOE2(158)APOE-/-LDLR-/-. No differences were observed in the areas of the atherosclerotic lesions between the three groups of mice. In addition, no differences were found in the cellular composition of these lesions. The pathological evaluation is described in the following table:

E3Leiden E-/- LDLR-/- E2(158) E-/- LDLR-/- E-/- LDLR-/-
Myocardial degeneration + - +
Pericentral hepatocellular lipid deposition +++ - +
Xanthomas (typical) + + ++
Glomerulosclerosis ++ + +

The extent of the xanthomatous lesions in the APOE-/- LDLR-/- mice was significantly increased as compared to the APOE3Leiden and APOE2(158) E-/- LDLR-/- mice. APOE2(158) E-/- LDLR-/- have less severe foamy cell infiltrations in heart and kidney, and no lipid depositions in the liver in comparison with the two other lines. Thus, under these circumstances, apoE2 can rescue lesion formation in the tissues but not the vessel wall.

Van Vlijmen BJM, K Willems van Dijk, HB van t Hof, PJJ van Gorp, H Van der Boom. ML Breumer, MH Hofker, LM Havekes. In the absence of endogenous mouse apolipoprotein E, apolipoprotein E*2 (Arg 158®Cys) transgenic mice develop more severe hyperproteinemia than apolipoprotein E*3-Leiden transgenic mice. J Biol Chem 1996;271:30595.

Van Vlijmen BJM, AMJM van den Maagdenberg, MJJ Gijbels H van der Boom, H HogenEsch, RR Frants, MH Hofker, LM Havekes. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J Clin Invest 1994;93:1403.

Richard S. Smith, M.D., D.Med.Sci.
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500
Rss@aretha.jax.org, 207-288-6283, fax 207-288-6276

Understanding the anatomy and embryology of the mouse eye is essential for evaluation of histopathologic findings. Removal of the eye, fixation, and sectioning require specialized techniques to avoid inducing artifacts that produce incorrect conclusions. Since the eye is an asymmetric structure with regional variations in anatomy, careful attention to plane of section is critical for correct interpretation. The structure of the eye and the methodology necessary to translate that structure to a slide will be reviewed. The unique techniques available for producing clinical photographs of ocular structures are an important adjunct to microscopic evaluation.
Specialized techniques useful in other tissues are usually easily adapted to the eye. Other speakers will review many of these, so discussion will be limited to some adaptations made in our laboratory for ultrastructural, immunohistochemical, and cell cycle applications.
Time constraints limit discussion to three topics:
(1) Glaucoma occurs in aging inbred 1 and knockout mice and demonstrates the same histopathological changes as human glaucoma. Recent work has revealed that glaucoma severity is modified by the presence of other genes. (Chang, B., et al., in press) A transgenic mouse with the human congenital glaucoma gene,2 Cyp1B1, demonstrates anatomic changes similar to those seen in human disease, confirming the utility of such an approach to generate mouse models (John, S., Gonzalez, F., and Smith, R., unpublished observations)
(2) Genes that control lens morphogenesis 3-7 often affect other aspects of ocular development, including induction of microphthalmia. The investigator must be aware of the effects of background, since certain strains of mice have a variable incidence of microphthalmia/cataracts 8 that is increased by external stimuli. 9
(3) The retina is capable of a limited response to genetic stimuli and findings
such as retinal dysplasia exist in many induced mutations as a common
epiphenomenon.10-11 The effects of genetic background can produce prominent
differences in retinal phenotype.12
References
1. John SWMJ, Smith RS, Savinova O, et al. Essential iris Atrophy, pigment dispersion and glaucoma in DBA/2J mice. Invest. Ophthalmol. Vis. Sci. 1998;39:951-62.
2. Bejjani BA, Lewis RA, Tomey KF, et al. Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant cause of primary congenital glaucoma in Saudi Arabia. Am. J. Hum. Genet. 1998;62:325-33.
3. Smith RS, Sundberg JP, Linder CC. Mouse mutations as models for studying cataracts. Pathobiology. 1997;65:146-54.
4. Robinson ML, Ohtaka-Maruyama C, Chan CC, et al. Disregulation of ocular morphogenesis by lens-specific nexpression of FGF-3/Int-2 in transgenic mice. Devel. Biol. 1998;198:13-31.
5. Dahl E, Koseki H, Balling R. Pax genes and organogenesis. Bioessays. 1997;19:755-65.
6. Egwuagu CE, Sztein J, Chan CC, Mahdi R, et al. gInterferon expression disrupts lens and retinal differentiation in transgenic mice. Devel. Biol. 1994;166:557-68.
7. Gotz W. Transgenic models for eye malformations. Ophthalmic Genet. 1995;16:85-104.
8. Smith RS, Roderick TH, Sundberg JP. Microphthalmia and associated abnormalities in inbred black mice. Lab. Animal Med. 1994;44:551-60.
9. Cook CS, Nowotney AZ, Sulik KK. Fetal alcohol syndrome: eye malformations in a mouse model. Arch. Ophthalmol. 1987;105:1576-1581.
10. Omri B, Blancher C, Neron B, et al. Retinal dysplasia in mice lacking p56lck. Oncogene. 1998;16:2351-56.
11. Humphries MM, Rancourt D, Farrar GJ, et al. Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat. Genet. 1997;15:216-19.
12. Ikeda, S, Hawes, NL, Chang, B., Avery, CS, Smith, RS, Nishina, PM Ocular abnormalities in C57BL/6 but not 129/Sv p53 deficient mice. In press.

Tracie E. Bunton, D.V.M., Ph.D.
Associate Professor, Division of Comparative Medicine, School of Medicine, The Johns Hopkins University, 456 Ross Research Building, 720 Rutland Avenue, Baltimore MD 21205
Tebunton@welchlink.welch.jhu.edu, 410-955-3273, Fax 410-550-5068

Genetic knockout mice are developed to study gene function and the pathogenesis of disease in human genetic disorders. Cardiovascular organogenesis involves complex prenatal and postnatal morphogenetic events which when altered may result in sequellae ranging from embryo lethality to
postnatal structural and functional deficits. The analysis of phenotype and determination of pathogenesis in these models presents a daunting challenge for the pathologist. Two mouse knockout models with different cardiovascular syndromes will be used to illustrate some of the problems encountered. The TGF b superfamily is implicated in cardiac malformations related to left-right cardiac looping and myogenesis. In a TGF b knockout mouse we have seen a range of defects including situs inversus, atrial and ventricular septal defects, double outlet right ventricle, and persistent left cranial vena cava. Marfan Syndrome is a connective tissue disorder of fibrillin-1 characterized by cardiovascular disease and ocular, skeletal, and skin manifestations. In two separate fibrillin-1 knockout mouse models we have identified severe aortic lesions with aneurysm, and skeletal abnormalities. In describing the phenotypes of these models, the morphologic techniques used to problem solve will be presented.

Saltis J, Agrotis A, Bobik A: Regulation and interactions of transforming growth factor-b with cardiovascular cells: Implications for development and disease. Clinical and Experimental Pharmacology and Physiology 23:193-200, 1996.

Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Biery NJ, Bunton TE, Dietz HC, Ramierz F. Targeting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nature Genetics 17:218-222, 1997

Michael Eckhaus, V.M.D.
Building 28A, Room 115
NIH, Bethesda, MD 20892
neckhaus@vrp.ncrr.nih.gov, 301-496-4465, fax 301-402-1068

The body of knowledge related to renal pathology in genetically engineered mice (GEM) has expanded greatly during the past several years. Selected transgenic and knock-out murine models of renal disease will be reviewed. The phenotypic spectrum of renal lesions in GEM, including developmental abnormalities, polycystic disease, and progressive glomerular disease will be discussed. The role of promoters, homeobox genes, hormones, oncogenes, viral genes, and immune-mediators as related to renal disease morphology will be reviewed.

Grandaliano G, Choudhury G and Abboud H: Transgenic animal models as a tool in the diagnosis of kidney diseases. Sem Nephrol 15: 43-49, 1995

Kone B: Molecular approaches to renal physiology and therapeutics. Sem Nephrol 18: 102-121, 1998

Kopp JB: Gene expression in kidney using transgenic approaches. Exp Nephrol 5:157-167, 1997

Kopp JB and Klotman PF: Transgenic animal models of renal disease and pathogenesis. Am J Physiol 269(5Pt2): F601-F620, 1995

Matsusaka T and Ichikawa I: Gene targeting in nephrology. Exp Nephrol 5:168-173, 1997

Zhang Z, Kundu GC, Yuan CJ, Ward JM, Lee EJ, DeMayo F, Westphal H, Mukherjee AB. Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin.
Science 276:1408-12, 1997.

Ryffel B: Gene knockout mice as investigative tools in pathophysiology. Int J Exp Pathol 77:125- 141, 1996

Saito A, Yamazaki H, Nakagawa Y and Arakawa M: Molecular genetic of renal diseases. Int Med 36: 81-86, 1997

Schieren G, Pey R, Bach J, Hafner M and Gretz N: Murine models of polycystic kidney disease. Nephrol Dial Transplant 11(Suppl 6): 38-45, 1996

Striker GE, He C, Liu Z, Yang D, Zalups R, Esposito C and Striker LJ: Pathogenesis of nonimmune glomerulosclerosis: Studies in animals and potential applications to humans. Lab Invest 73: 596-605, 1995

James G. Fox, D.V.M. and David B. Schauer, D.V.M., Ph.D., Charles A. Dangler, D.V.M., Ph.D.
Division of Comparative Medicine, Massachusetts Institute of Technology, 37 Vassar Street, Building 45, Cambridge, MA 02139-4307, Phone: 617-253-1757, Fax: 617-258-5708, Email: jgfox@mit.edu, cdangler@mit.edu

The human inflammatory bowel diseases (IBD's), Crohn's disease and ulcerative colitis, are multifactorial disorders whose etiolgy remains unknown. Numerous mouse models have been developed to help understand the complex interaction between the immune system and intestinal antigens - particularly bacteria - which appear to be involved in the pathogenesis of disease. For example, when IL-2 deficient mice are rederived by hysterectomy and maintained under specific-pathogen-free (SPF) or germfree conditions, they exhibit no clinical signs of disease. The SPF animals do have mild histopathological changes, but lesions are completely absent from the colonic tissue of germfree animals, up to 20 weeks of age. In each report of the original descriptions of IBD in knockout mice, animals were examined for known pathogenic agents. The animals were reported to be free from known pathogens. However, at the time these studies were conducted Helicobacter spp. were not recognized as murine pathogens. Infection with the mouse pathogen Helicobacter hepaticus is now associated with disease in mice which are genetically and/or immunologically predisposed to the development of IBD. H. hepaticus, like the human gastroduodenal pathogen H. pylori, expresses putative virulence factors which may contribute to chronic mucosal inflammation, epithelial cell proliferation, and an increased risk for cancer. Both H. hepaticus and H. bilis, another murine helicobacter, cause marked inflammation of the lower bowel when inoculated into defined microbiota scid mice. Recently IL-10 deficient mice, when maintained under controlled microbial conditions and inoculated with H. hepaticus, developed severe typhlocolitis. Additional novel urease negative Helicobacter spp. can also have a similar proinflammatory stimulus of the large intestine in various strains of mice. Further studies designed to characterize the mechanism by which infection with specified bacterial species (i.e. enterohepatic Helicobacter spp.) can affect expression of disease in mouse models of IBD will aid in our understanding of this complex disease process. It is hoped that these studies will lead to the development of rational therapy for patients suffering from Crohn's disease and ulcerative colitis.

Berg, O. J., N. Davidson, R. Kuhn, W. Muller, S. Menon, G. Holland, and L. Thompson-Snipes. 1996. Enterocolitis and colon cancer in interleukin 10 deficient mice are associated with aberrant cyotokine production and CD4 + Th1- like response. J Clin Invest 98:1010-1020.
Cahill, R. J., C. J. Foltz, J. G. Fox, C. A. Dangler, F. Powrie, and D. B. Schauer. 1997. Inflammatory bowel disease: an immune mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infect Immun 65:3126-3131.
Foltz CJ, Fox JG, Cahill R, Murphy JC, Yan L, Shames B, Schauer DB. Spontaneous inflammatory bowel disease in multiple mutant mouse lines: association with colonization by Helicobacter hepaticus. Helicobacter 3:69-78, 1998.
Foltz, C., J. G. Fox, L. Yan, and B. Shames. 1995. Evaluation of antibiotic therapies for the eradication of Helicobacter hepaticus. Antimicrob Agents Chemother 36:1292-1294.
Fox, J. G., F. E. Dewhirst, J. G. Tully, B. J. Paster, L. Yan, N. S. Taylor, M. J. Collins, P. L. Gorelick, and J. M. Ward. 1994. Helicobacter hepaticus sp. nov, a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 32:1238-1245.
Fox, J. G., L. Yan, B. Shames, J. Campbell, J. C. Murphy, and X. Li. 1996. Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect Immun 64:3673-3681.
Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263-274.
Kullberg MC, Ward JM, Gorelick PL, Caspar P, Hieny S, Cheever A, Jankovic D, Sher A.
Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism. Infect Immun 66:5157-66, 1998.
Powrie, F., M. Leach, S. Mauze, S. Menon, L. Caddle, and R. Coffman. 1994. Inhibition of Th1 response prevents inflammatory bowel disease in scid mice reconstitued with CD45RB hi CD4+T cells. Immunity 1:553-562.
Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, and I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253-261.
Shames, B., J. G. Fox, F. E. Dewhirst, L. Yan, Z. Shen, and N. S. Taylor. 1995. Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay. J Clin Microbiol 33:2968-2972.
Shomer, N. H., C. A. Dangler, and J. G. Fox. 1997. Helicobacter bilis induced inflammatory bowel disease (IBD) in defined flora scid mice. Infect Immun 65:4858-4864.
Ward, J. M., M. R. Anver, D. C. Haines, and R. E. Benveniste. 1994. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol 145:959-968.
Ward, J. M., M. R. Anver, D. C. Haines, J. M. Melhorn, P. Gorelick, L. Yan, and J. G. Fox. 1996. Inflammatory large bowel disease in immunodeficient mice naturally infected with Helicobacter hepaticus. Lab Anim Sci 46:15-20.