Faculty Bibliography

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages, but rarely generate extra-embryonic cell types. We show that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells to yield both embryonic and extra-embryonic lineages and strongly induce MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with totipotent 2-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and represses MERVL expression through transcriptional regulation, at least in part, by targeting the transcription factor Gata2. Altogether, our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential, raising the tantalizing possibility of culturing bi-potential ESCs to explore the molecular basis for totipotency.

In this study, we outline a regulatory network that involves the p53 tumor suppressor family and the Wnt pathway acting together with the TGF-beta pathway in mesendodermal differentiation of mouse and human embryonic stem cells. Knockout of all three members, p53, p63, and p73, shows that the p53 family is essential for mesendoderm specification during exit from pluripotency in embryos and in culture. Wnt3 and its receptor Fzd1 are direct p53 family target genes in this context, and induction of Wnt signaling by p53 is critical for activation of mesendodermal differentiation genes. Globally, Wnt3-activated Tcf3 and nodal-activated Smad2/3 transcription factors depend on each other for co-occupancy of target enhancers associated with key differentiation loci. Our results therefore highlight an unanticipated role for p53 family proteins in a regulatory network that integrates essential Wnt-Tcf and nodal-Smad inputs in a selective and interdependent way to drive mesendodermal differentiation of pluripotent cells.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2-/- mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2loxP/loxP mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.

In October 2013, the International Combined Orthopaedic Research Societies (ICORS; https://urldefense.proofpoint.com/v2/url?u=http-3A__i- 2Dcors.org&d=DQIBAg&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=KRXeNoRy5_8lkSwAJ G5vjS1yT0aFSItfe494dmkdSVs&m=Q128C029R6hKpAWrbdSw1X1eyRa_A15Le8lQ5N7b464&s=WM_- 4Atc-eAEEnVXWhP05NYU50wYQsZ1AXGjxxkozSw&e= ) was founded with inaugural member organisations from the previous Combined Orthopaedic Research Society, which had sponsored combined meetings for more than 2 decades. The ICORS is dedicated to the stimulation of orthopaedic and musculoskeletal research in fields such as biomedical engineering, biology, chemistry, and veterinary and human clinical research. The ICORS seeks to facilitate communication with member organisations to enhance international research collaborations and to promote the development of new international orthopaedic and musculoskeletal research organisations. Through new categories of membership, the ICORS represents the broadest coalition of orthopaedic research organisations globally.

One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling.