Geminin Promotes an Epithelial-to-Mesenchymal Transition in an Embryonic Stem Cell Model of Gastrulation

Geminin is a nuclear protein that performs the related functions of modulating cell cycle progression by binding Cdt1, and controlling differentiation by binding transcription factors. Since embryonic stem cells (ESC) and the epiblast share a similar gene expression profile and an attenuated cell cycle, ESC form an accessible and tractable model system to study lineage choice at gastrulation. We derived several ESC lines in which Geminin can be inducibly expressed, and employed short hairpin RNAs targeting Geminin. As in the embryo, a lack of Geminin protein resulted in DNA damage and cell death. In monolayer culture, in defined medium, Geminin supported neural differentiation; however, in three-dimensional culture, overexpression of Geminin promoted mesendodermal differentiation and epithelial-to-mesenchymal transition. In vitro, ESC overexpressing Geminin rapidly recolonized a wound, downregulated E-cadherin expression, and activated Wnt signaling. We suggest that Geminin may promote differentiation via binding Groucho/TLE proteins and upregulating canonical Wnt signaling.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140196/1/scd.2012.0050.pd

Expression of Neurogenin 1 in Mouse Embryonic Stem Cells Directs the Differentiation of Neuronal Precursors and Identifies Unique Patterns of Down‐stream Gene Expression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96662/1/dvdy23920.pd

Ultrastructural analysis of chromosome translocation-induced neurol tube defects

Neurol tube closure defects occurred in 33% of the embryos obtained from matings of male mice heterozygous for a reciprocal chromosome translocation (T(2;4)lSn) with normal female CFLP mice. Light and electron microscopic observations of neuroepithelium and mesenchyme in affected embryos indicated two distinct types of anomalies occurred. The first consisted of neuroepithelial hypertrophy and neural tube closure defects. These defects most frequently affected the midbrain and hindbrain, but occasional defects of the lumbosacral neural tube were also observed. Unlike the highly organized, pseudostratified neuroepithelium in control embryos, neuroepithelial cells became stratified and formed cell islands with secondary lumina within the wall of the neural tube. The second condition was associated with a reduction in neuroepithelial thickness, considerable neuroepithelial and neural crest cell death, basal lamina alterations and premature invasion of the neuroepithelium by subjacent endothelial cells. In both cases, the cephalic mesenchyme cells, rather than their normal stellate appearance, were markedly elongated in shape and reduced in area. The number of cell-cell contacts between mesenchymal cells was also reduced significantly. These results are discussed in light of recent theories regarding the role of mesenchyme and extracellular matrix in neurulation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26162/1/0000239.pd

The 2002 Leon and Josephine Winkelman Lecture, University of Michigan School of Social Work

The Leon and Josephine Winkelman Memorial Lecture was established at the U-M School of Social Work by the Winkelman brothers (Stanley J., John H., Frederick R., and Henry R.) as a memorial to their parents. The lecture provides a forum for presenting new and emerging knowledge from the social sciences and helping professions, and discussion of the application of that knowledge to the development of social policy, the organization and management of social welfare services, and the delivery of social work services. The selection of topics and scholars reflects the interdisciplinary character of the lecture. This is in keeping with the representation of several disciplines in the Social Work faculty, the School's links with social sciences through its interdisciplinary Joint Doctoral Program in Social Work and Social Science, and the School's collaborations with the School of Public Health, the Medical Center, and the Institute of Gerontology.The Leon and Josephine Winkelman Family; School of Social Work; alumni, faculty, and friends of the School of Social Workhttp://deepblue.lib.umich.edu/bitstream/2027.42/49497/3/2002 Winkelman Lecture OShea.pd

Expression of thrombospondin in the adult nervous system

Thrombospondin (TSP) is an extracellular matrix molecule that has been previously associated with neural development and neurite outgrowth in vitro. Little is known, however, about the expression of TSP in the adult nervous system. In this study, TSP localization was examined in nervous tissue from adult mouse, goldfish, newt, and adult and juvenile Xenopus. TSP was associated with neurons in the brains of all species examined. TSP was present in central nerve tracts capable of regeneration, such as the goldfish, Xenopus, and newt optic nerves, but was absent from tracts not capable of regeneration, such as the mouse optic nerve. TSP was also present in the neuropil of goldfish and newt spinal cord, but was restricted to motor neurons in mice and adult Xenopus. In addition, TSP was observed in sciatic nerves of mice, Xenopus, and newt. These results indicate a correlation between the presence of TSP and the potential for successful nerve regeneration across a wide range of animal classes. © Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50059/1/903400109_ftp.pd

Basal lamina and extracellular matrix alterations in the caudal neural tube of the delayed Splotch embryo

Regional patterns of deposition of laminin (LN), fibronectin (FN), type IV collagen (IV), and heparan sulfate proteoglycan (HSPG) were examined during the formation of the caudal neural tube in embryos homozygous for the delayed Splotch gene and in their normal littermates. Delayed Splotch embryos had neural tube closure defects which extended from the posterior neuropore into the region formed by secondary neurulation. During posterior neuropore closure these components were normally restricted to forming basal laminae, with FN and HSPG additionally deposited in the mesenchyme. Unlike control embryos in which medial regions of the neuroepithelial basal lamina contained greatest amounts of all four, the dorsolateral zone contained less LN and IV and more FN and HSPG, in affected embryos these components were less densely deposited medially, reflecting perhaps the poor structural organization of the notochord. The neuroepithelial basal lamina was often disorganized and wavy compared to the linear pattern typical of controls. By the 12th day, the posterior neuropore of controls had closed and secondary neurulation was underway; however in delayed Splotch embryos, the neural folds remained widely splayed and epithelium newly formed via secondary neurulation extended that abnormally open configuration to the tip of the tailbud. In controls, with mesenchymal cell aggregation FN and HSPG were displaced from between cells to the forming basal lamina. As a central lumen formed within the aggregate LN and IV were added to the basal lamina, and the newly formed epithelium merged with the anterior neural tube. In delayed Splotch embryos, FN and HSPG were incompletely removed from aggregating cell surfaces, the normal morphogenetic cell shaping changes failed to occur and in many embryos a central lumen did not form; the overgrown, aggregated cells merging with the abnormally splayed anterior neural folds. In addition, the critical enrichment of FN and HSPG present between newly formed and consolidated neuroepithelium was displaced in delayed Splotch embryos.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26469/1/0000004.pd

Application of frozen thin sectioning immunogold staining to the study of the developing neuroepithelial basal lamina

In order to examine the deposition of basal lamina components in the developing neurocpithelium, a technique for frozen thin sectioning and immunogold staining of early embryonic tissue was developed. Different fixatives and buffer systems were evaluated to determine which best retained immunoreactivity and satisfactory ultrastructure of day 9 and 10 mouse embryos. Fixation in sodium phosphate and sodium bicarbonate buffers did not retain antigenicity, and incubations in TBS ( Tris hydroxymethyl-aminomethane buffered saline) in an effort to ‘restore’ immunoreactivity were similarly unsuccessful. Fixation in sodium cacodylate buffer, however, did retain the antigenicity of basal lamina components; the pattern of type IV collagen and laminin distribution was clearly determined. These results represent the first report of on-grid immunocytochemistry of carly embryonic material.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47407/1/418_2004_Article_BF00518729.pd

Transplacental RNAi: Deciphering Gene Function in the Postimplantation-Staged Embryo

RNAi offers the opportunity to examine the role in postimplantation development of genes that cause preimplantation lethality and to create allelic series of targeted embryos. We have delivered constituitively expressed short hairpin (sh) RNAs to pregnant mice during the early postimplantation period of development and observed gene knockdown and defects that phenocopy the null embryo. We have silenced genes that have not yet been “knocked out” in the mouse (geminin and Wnt8b), those required during earlier cleavage stages of development (nanog), and genes required at implantation (Bmp4, Bmp7) singly and in combination (Bmp4 + Bmp7), and obtained unique phenotypes. We have also determined a role in postimplantation development of two transcripts identified in a differential display RT-PCR screen of genes induced in ES cells by noggin exposure, Aggf1 and an Est (GenBank AK008955). Systemic delivery of shRNAs provides a valuable approach to gene silencing in the embryo

Geminin Is Required for Epithelial to Mesenchymal Transition at Gastrulation

Geminin is a multifunctional protein previously suggested to both maintain the bone morphogenetic protein inhibition required for neural induction and to control cell-cycle progression and cell fate in the early embryo. Since Geminin is required in the blastocyst on E3.5, we employed shRNA to examine its role during postimplantation development. Geminin knockdown inhibited the epithelial to mesenchymal transition (EMT) required at gastrulation and neural crest delamination, resulting in anterior-posterior axis and patterning defects, while overexpression promoted EMT at both locations. Geminin was negatively correlated with expression of E-cadherin, which is critically involved in controlling epithelial architecture. In addition, Geminin expression level was correlated with Wnt signaling and expression of the Wnt target gene Axin2 and with Msx2, and negatively correlated with the expression of Bmp4 and Neurog1 in quantitative reverse transcriptase?polymerase chain reaction analysis of RNAs from individual embryos. These results suggest that in addition to patterning the early embryo, Geminin plays a previously unrecognized role in EMT via its ability to affect Wnt signaling and E-cadherin expression.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98475/1/scd%2E2011%2E0483.pd

Analysis of the Factors that Limit the Ability of Feeder Cells to Maintain the Undifferentiated State of Human Embryonic Stem Cells

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78130/1/scd.2008.0010.pd