Gametogenesis is the process whereby a series of complex developmental events give rise to a highly specialized cell type, the gamete, which is specifically designed to deliver a haploid genome and to support the early development of the embryo. In multi-cellular organisms, germ cell development is orchestrated by complex interactions between the developing germ cells and their dynamic somatic environment; when these events are disrupted, infertility and aneuploidy result. Therefore, to understand human reproduction, it is critical to both identify and understand the molecular mechanisms that regulate germ cell development. Our research relies primarily on functional genomics to identify the genes required for mammalian reproduction, using the laboratory mouse as a model. As part of the Genetic Resource Science team, we also develop and/or implement new tools and technologies for functional genomics, including high throughput sequencing approaches and new ES cell resources.
Germ cell development
My research focuses on the molecular pathways governing the development of germ cells in the mammalian embryo and in the adult gonad. I am particularly interested in the regulation of germ cell division during the proliferative stages of development and during meiosis. Current projects include the cloning and characterization of two recessive, induced mutants (gcd2 and repro7), which cause infertility via disruption of germ cell proliferation and/or meiosis in both the male and female germline. Using high throughput re-sequencing, I have now successfully identified the causative mutations in these mutants and am currently using these data to tease out the underlying mechanisms by which these mutations contribute to infertility.
Germ cell depletion 2 (gcd2) is an EMS induced mutation that causes male and female infertility due to depletion of primordial germ cells during development. The mutation was mapped to a region of less than 3 Mb on Chr 2 (Reinholdt et al., 2008) and targeted, high throughput re-sequencing revealed a missense mutation in the gene Kif18a (unpublished). Kif18a encodes a kinesin protein that has been previously shown to localize to the mitotic spindle during mitosis and to the cytoplasm during interphase. The protein has been implicated in proper maintenance of the mitotic spindle and also in cytoplasmic trafficking in response to signaling through the estrogen receptor. However, a role for this protein in primordial germ cell development has yet to be established. I am currently using a variety of approaches to examine the role of this gene in primordial germ cell development using both mice and zebrafish. Taking advantage of my previous data on strain background effects (Reinholdt et al, 2008), I am also embarking on a genetic screen to map modifier loci.
The repro7 mutation is an ENU induced autosomal recessive mutation causing infertility in both male and female mice. Analysis of prepubertal repro7/repro7 mice showed that defects in spermatogenesis are first apparent at 17 dpp, when the majority of spermatoyctes are in the pachytene stage of meiotic prophase. In repro7/repro7 female mice, the majority of oocytes are lost between E17 and 1dpp, again consistent with defects in germ cell development at the pachytene stage of meiosis. I have sequenced the entire candidate region by high throughput sequencing and analysis of the data is currently underway.
Mutagenesis and genomics
Unbiased approaches are the mainstay of forward genetic approaches for functional annotation of genes. These phenotype driven approaches involve the use of spontaneous and induced mouse mutants with clinically relevant phenotypes and subsequent molecular cloning of the causative lesions / genes underlying those phenotypes. Gene identification has been the historical bottleneck in this process, however the recent advent of high throughput sequencing has removed this bottleneck, resulting in a resurgence of unbiased, phenotype driven approaches to gene discovery. In addition to creating new ENU mutations via our spontaneous mouse mutant program and via saturation mutagenesis, I am currently developing high throughput sequencing pipelines for mutation discovery via targeted re-sequencing of contiguous regions (D'Ascenzo et al., 2009), whole exomes or whole genome re-sequencing. I am also developing high throughput sequencing approaches to identify translocation breakpoints and transgene insertion sites in the mouse genome. Current projects include the identification of the translocation breakpoint in a mouse model of Down syndrome, Ts65Dn.
ES cell biology
Recent developments in our understanding of pluripotency and strain background effects have allowed my laboratory to implement highly efficient generation of pluripotent embryonic stem cells (ES cells) from a variety of strain backgrounds, including strains that have proven recalcitrant to such efforts in the past. I am now funded to derive new ES cell lines from disease models (Ts65Dn), tool strains (Hlbx-9 GFP), and inbred strains, like DBA/2J and NOD.
We actively engage in collaborations and are always seeking new projects, especially those that align with our current research interests and resource development goals. Please don't hesitate to contact me with questions or ideas.
Principal Investigator: Laura Reinholdt, Ph.D.
Research Assistant I: Candice Byers, Ian Greenstein
Research Assistant II: Anne Czechanski
Executive Assistant: Aimée Picard
Reinholdt LG, Howell GR, Czechanski AM, Macalinao DG, Macnicoll KH, Lin CS, Donahue LR, John SW. 2012. Generating embryonic stem cells from the inbred mouse strain DBA/2J, a model of glaucoma and other complex diseases. PLoS One. 2012;7(11):e50081. doi: 10.1371/journal.pone.0050081. PMCID: PMC3507949
Simon MM, Mallon AM, Howell GR, Reinholdt LG. 2012. High throughput sequencing approaches to mutation discovery in the mouse. 2012 Sep 19. Mamm Gen. Epub ahead of print, DOI 10.1007/s00335-012-9424-0.
Wong K, Bumpstead S, van der Weyden L, Reinholdt LG, Wilming L, Adams DJ, Keane TM. 2012. Sequencing and characterization of the FVB/NJ mouse genome. Genome Biol. 2012 Aug 23;13(8):R72. PMCID: PMC3491372
Davisson MT, Bergstrom DE, Reinholdt LG, Donahue LR. Discovery Genetics: The History and Future of Spontaneous Mutation Research, Curr Protocol Mouse Biol. 2012; 2:103-118.
Fritsche M, Reinholdt LG, Lessard M, Handel MA, Bewersdorf J, Heermann DW. 2012. The Impact of Entropy on the Spatial Organization of Synaptonemal Complexes within the Cell Nucleus. PLoS One 2012; 7(5):e36282. PMCID: PMC3344857
Reinholdt LG, Ding Y, Gilbert GT, Czechanski A, Solzak JP, Roper RJ, Johnson MT, Donahue LR, Lutz C, Davisson MT. 2011. Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn. Mamm Genome, 2011; 22(11-12):685-91. PMCID: PMC3505986
Keane TM, Goodstadt L, Dancek P, White MA, Wong K, Yalcin B, Heger A, Agam A, Slater G, Goodson M, Furlotte NA, Eskin E, Nellaker C, Whitley H, Cleak J, Janowitz D, Hernandez-Pliego P, Edwards A, Belgard TG, Oliver, P, McIntyre RE, Bhomra A, Nicod J, Gan X, Yuan W, van der Weyden L, Steward CA, Bala S, Stalker J, Mott R, Durbin R, Jackson IJ, Czechanski A, Guerra-Assuancao JA, Donahue LR, Reinholdt LG, Payseur BA, Ponting CP, Birney W, Flint J, Adams DJ. 2011. Mouse genomic variation and its effect on phenotypes and gene regulation. Nature 477, 289-294. PMCID: PMC3276836
Fairfield H, Gilbert GJ, Barter M, Corrigan RR, Curtain M, Ding Y, D'Ascenzo M, Gerhardt DJ, He C, Huang W, Richmond T, Rowe L, Probst FJ, Bergstrom DE, Murray SA, Bult C, Richardson J, Kile B, Gut I, Hager J, Sigurdsson S, Mauceli E, Di Palma F, Lindblad-Toh K, Cunningham ML, Cox TC, Justice MJ, Spector MS, Lowe SW, Albert T, Donahue LR, Jeddeloh J, Shendure J, Reinholdt LG. 2011. Mutation discovery in mice by whole exome sequencing. Genome Biol. 12(9):R86. PMID: 21917142.Sinnar SA, Small CL, Evanoff RM, Reinholdt LG, Griswold MD, Kopito RR, Ryu KY. 2011. Altered testicular gene expression patterns in mice lacking the polyubiquitin gene Ubb. Mol Reprod Dev. 78(6):415-25. PMCID: PMC3133727. Beamer WG, Shultz KL, Coombs HF 3rd, Demambro VE, Reinholdt LG, Ackert-Bicknell CL, Canalis E, Rosen CJ, Donahue LR. 2011. BMD regulation on mouse distal Chromosome 1, candidate genes, and response to ovariectomy or dietary fat. J Bone Miner Res, 26(1):88-99. PMCID: PMC3179313
Wu B, Potter CS, Silva KA, Liang Y, Reinholdt LG, Alley LM, Rowe LB, Roopenian DC, Awgulewitsch A, Sundberg JP. 2010. Mutations in Sterol O-Acyltransferase 1 (Soat1) Result in Hair Interior Defects in AKR/J Mice. J Invest Dermatol. [Epub ahead of print]. PMCID: PMC2955156.
Reinholdt LG, Czechanski A, Kamdar S, King BL, Sun F, Handel MA. 2009. Meiotic behavior of aneuploid chromatin in mouse models of Down syndrome. Chromosoma 118:723-36. PMCID: PMC2848991.
D'Ascenzo M, Meacham C, Kitzman J, Middle C, Knight J, Winer R, Kukricar M, Richmond T, Albert TJ, Czechanski A, Donahue LR, Affourtit J, Jeddeloh JA, Reinholdt L. 2009. Mutation discovery in the mouse using genetically guided array capture and resequencing. Mamm Genome 20:424-36. PMCID: PMC2829192.
Ryu KY, Sinnar SA, Reinholdt LG, Vaccari S, Hall S, Garcia MA, Zaitseva TS, Bouley DM, Boekelheide K, Handel MA, Conti M, Kopito RR. 2008. The mouse polyubiquitin gene bb is essential for meiotic progression. Mol Cell Biol 28(3):1136-46. PMCID: PMC2223379.
Ward JO, Reinholdt LG, Motley WW, Niswander LM, Deacon DC, Langlais K, Backus VL, Schimenti KJ, O'Brien MJ, Eppig JJ, Schimenti JC. 2007. Mutation in mouse Hei10, an E3 ubiquitin ligase, disrupts meiotic crossing over. PLoS Genet 3(8):e139.
Reinholdt LG, Munroe RJ, Schimenti JC. 2006. The induced mutation, gcd2, causes depletion of primordial germ cells in the mouse embryo. Mech Dev 123(7):559-69.
Handel MA, Lessard C, Reinholdt LG, Schimenti JC, Eppig JJ. 2006. Mutagenesis as an unbiased approach to identify novel contraceptive targets. Mol Cell Endocrinol 250(1-2):201-5.
Reinholdt LG, Schimenti JC. 2005. Mei1 is epistatic to Dmc1 during mouse meiosis. Chromosoma 114(2):127-34.
Reinholdt LG, Ashley T, Schimenti JC, Shima N. 2004. Forward genetic screens for meiotic and mitotic recombination-defective mutants in mice. Methods Mol Biol 262:87-107.
Bannister LA, Reinholdt LG*, Munroe RJ, Schimenti JC. 2004. Positional cloning and characterization of mei8, a disrupted allele of Rec8. Genesis 40(3):184-94. *equal contribution
Ward JO, Reinholdt LG, Hartford SA, Wilson LA, Munroe RJ, Schimenti KJ, Libby BJ, O'Brien M, Pendola JK, Eppig J, Schimenti JC. 2003. Toward the genetics of mammalian reproduction: induction and mapping of gametogenesis mutants in mice. Biol Reprod 69:1615-1625.
Libby BJ, Reinholdt LG, Schimenti JC. 2003. Positional cloning and characterization of Mei1, a novel, vertebrate-specific gene required for meiotic chromosome synapsis in mice. Proc Natl Acad Sci USA 100(26):15706-11.
Reinholdt LG, Gutierrez GM, Krider HM. 2002. Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth, Lymantria dispar, is preceded by an aberrant prophase I. Chromosoma 111(3):136-46.
Garvey LK, Gutierrez GM, Krider HM. 2000. Ultrastructure and morphogenesis of the apyrene and eupyrene spermatozoa in the Gypsy Moth, Lymantria dispar (Lepidoptera: Lymnatriidae. Ann Entomol Soc Am 93(5):1147-1155