Nagy Lab projects

There are several areas of interest represented in the lab.

Vessel development

Our interest in vessel biology stems from targeting and characterizing the early developmental role of Vascular Endothelial Growth Factor-A (VEGF-A). Following the early role in promoting endothelial cell differentiation, the expression of VEGF-A remains under tight, cell type-specific control, implicating its function during the genesis of different organs as well. The early embryonic-lethal haplo-insufficient nature of VEGF-A made genetic studies rather difficult. To circumvent this limitation, we performed additional targeted alterations to VEGF-A, which resulted in a hypermorphic and a hypomorphic allele. These together with a Cre recombinase-conditional mutant (obtained from Napoleone Ferrara, Genentech), opened the possibility of genetic approaches for investigating the role of this growth factor in more detail, particularly during organogenesis. Currently, we have four organs in our focus; central nervous system, kidney yolk sac and bone.

VEGF heterozygous lethat phenotype
In our search for novel VEGF-like angiogenic factors, we are studying a new member of the PDGF superfamily. Although this new growth factor has significant homology to VEGF-A, it has turned out to be a new PDGF ligand (PDGF-C) with similar expression pattern as PDGF-A. PDGF-C deficiency in mice causes perinatal lethality and several developmental abnormalities.

 

Genomic imprinting

Genomic imprinting is a strong focus in the lab, it roots back to an early interest in parthenogenetic development. This pursuit has grown into an extensive genetic investigation of one of the most imprinted gene-populated regions on the mouse: distal chromosome 7. Using the paternally imprinted Mash2 gene, we have been gaining insight into the nature of the regulation of uniparental gene expression in this region.

from the mother
from the father

 

Embryonic Stem cells as a genetic model for the mouse


Several independent projects in my laboratory have converged and are now integrated into a complex genetic system completely built on Embryonic Stem Cells. This approach allows us to perform both forward and reverse genetics on the mouse genome, and efficiently address gene functions during development and/or disease without extensive animal breeding.


One of the most crucial parts of this complex is our newly developed ability to direct transgenic insertions to defined and pre-characterized site(s) in the genome, using a new integrase, PhiC31. This approach includes the design of a “docking site”, its placement into the genome and characterization of each integration site for expression permissiveness. Upon introduction of another specially designed, so-called “incoming” vector, the integrase catalyses the replacement of the docking site with the incoming sequence. With the published design, we were able to obtain a 90% efficiency for this cassette exchange. Several members of the lab are currently working on improving this efficiency and increasing it close to 100%.


A second pivotal component of the system is our new ES cell line derived from an 129xC57 F1 hybrid embryo, which showed superior developmental potential when aggregated with tetraploid embryos (resulting in completely ES cell-derived animals). Also, the genetically altered sublines retained this outstanding quality.


The third component is our ability to generate chromosome-specific homozygosity in ES cells. We have shown that the commonly used “high G418 selection” to obtain homozygous gene-knockout ES cell lines from heterozygous cells, preferentially creates homozygosity in the entire chromosome where the targeted locus was assigned. Furthermore, many aspects of the use of the Cre recombinase system for genome manipulation are well developed in the lab. Using these tools, a Cre recombinase-mediated creation of homozygosity is under development. This important tool will be integrated into a large-scale forward genetics project aiming to mutagenize the ES cell genome with chemical mutagens with subsequent creation of homozygosity in specific chromosomes. We expect this ES cell based system will raise the throughput and efficiency of recessive genetic screens in the mouse to at least one order of magnitude higher level then has previously been possible.

 

 

 

Stem cell biology

In the last five years, we have made a significant effort into understanding the differences in the efficiency of establishment and in vivo developmental potential of ES cells caused by their genetic background. The result of this large undertaking is that we have solved the problem of establishing ES cells from the “non obese diabetic” (NOD) mouse, which were known to have the most stubborn, non-permissive genetic background. However, in spite of the large number of cell lines tested, neither of them showed reasonable contribution to chimeras, nor did any of them result in germline transmission.
The lab is taking part in several Stem Cell Network (SCN) of Canada funded operations. One of our roles in their Stem Cell Plasticity program is to create and distribute ES cell and corresponding mouse “reagents” to other laboratories for cell plasticity studies.
We are also entering into the exciting field of human ES cell biology, with a near future plan of studying aspect of self-renewal and early differentiation processes. Our goal is to adapt the transgenic tools from our well-developed mouse system to the human ES cells.

  

 

Books or book chapters

We have always emphasized the dissemination of new methodologies and concepts in book chapters and reviews. The most comprehensive of all is the recently published 3rd edition of the “Manipulating the Mouse Embryo, A Laboratory Manual”. Three of the four authors are from our lab.

 

Collaborations

The leading philosophy of our research is to share information and reagents with others to help the progress of science with extreme openness.

  

 


Some relevant references from the lab

1. Tanaka, M. et al. Parental origin-specific expression of Mash2 is established at the time of implantation with its imprinting mechanism highly resistant to genome-wide demethylation. Mech Dev 87, 129-142 (1999).
2. Miquerol, L., Langille, B.L. & Nagy, A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development 127, 3941-3946 (2000).
3. Miquerol, L., Gertsenstein, M., Harpal, K., Rossant, J. & Nagy, A. Multiple developmental roles of VEGF suggested by a LacZ-tagged allele. Dev Biol 212, 307-322 (1999).
4. Damert, A., Miquerol, L., Gertsenstein, M., Risau, W. & Nagy, A. Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation. Development 129, 1881-1892 (2002).
5. Haigh, J.J. et al. Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling. Dev Biol 262, 225-241 (2003).
6. Eremina, V. et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111, 707-716 (2003).
7. Pinter, E., Haigh, J., Nagy, A. & Madri, J.A. Hyperglycemia-induced vasculopathy in the murine conceptus is mediated via reductions of VEGF-A expression and VEGF receptor activation. Am J Pathol 158, 1199-1206 (2001).
8. Ding, H. et al. The mouse Pdgfc gene: dynamic expression in embryonic tissues during organogenesis. Mech Dev 96, 209-213 (2000).
9. Nagy, A., Perrimon, N., Sandmeyer, S. & Plasterk, R. Tailoring the genome: the power of genetic approaches. Nat Genet 33 Suppl, 276-284 (2003).
10. Belteki, G., Gertsenstein, M., Ow, D.W. & Nagy, A. Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase. Nat Biotechnol 21, 321-324 (2003).
11. Lefebvre, L., Dionne, N., Karaskova, J., Squire, J.A. & Nagy, A. Selection for transgene homozygosity in embryonic stem cells results in extensive loss of heterozygosity. Nat Genet 27, 257-258 (2001).
12. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109 (2000).
13. Hadjantonakis, A.K., Macmaster, S. & Nagy, A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol 2, 11 (2002).
14. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev 76, 79-90 (1998).
15. Lobe, C.G. et al. Z/AP, a double reporter for cre-mediated recombination. Dev Biol 208, 281-292 (1999).
16. Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147-155 (2000).
17. Ohtsu, H. et al. Mice lacking histidine decarboxylase exhibit abnormal mast cells. FEBS Lett 502, 53-56 (2001).
18. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Non-invasive sexing of preimplantation stage mammalian embryos. Nat Genet 19, 220-222 (1998).
19. Hadjantonakis, A.K., Cox, L.L., Tam, P.P. & Nagy, A. An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta. Genesis 29, 133-140 (2001).
20. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo, A Laboratory Manual, Edn. 3rd. (Cold Spring Harbor Press, 2003).

 

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