Biomedical subjects

Suling Zhao

Publications and source records attributed to Suling Zhao.

5 recordsLinked to original sources

PubMed2005

Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development.

Recent studies of mouse mutant aphakia have implicated the homeobox gene Pitx3 in the survival of substantia nigra dopaminergic neurons, the degeneration of which causes Parkinson's disease. To directly investigate a role for Pitx3 in midbrain DA neuron development, we have analysed a line of Pitx3-null mice that also carry an eGFP reporter under the control of the endogenous Pitx3 promoter. We show that the lack of Pitx3 resulted in a loss of nascent substantia nigra dopaminergic neurons at the beginning of their final differentiation. Pitx3 deficiency also caused a loss of tyrosine hydroxylase (TH) expression specifically in the substantia nigra neurons. Therefore, our study provides the first direct evidence that the aphakia allele of Pitx3 is a hypomorph and that Pitx3 is required for the regulation of TH expression in midbrain dopaminergic neurons as well as the generation and/or maintenance of these cells. Furthermore, using the targeted GFP reporter as a midbrain dopaminergic lineage marker, we have identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain based on their temporal and topographical expression of Pitx3 and TH. Such an expression pattern may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3.

Animals↗

PubMed2004

SoxB transcription factors specify neuroectodermal lineage choice in ES cells.

Knowledge of lineage decision machinery in pluripotent embryonic stem (ES) cells may shed light on the process of germ layer segregation in the mammalian embryo and enable directed differentiation in vitro for biomedical applications. We have investigated the contribution of Class B1 Sox transcription factors to lineage choice during ES cell differentiation. We report that forced expression of Sox1 or Sox2 did not impair propagation of undifferentiated ES cells, but upon release from self-renewal promoted differentiation into neuroectoderm at the expense of mesoderm and endoderm. The efficient specification of a primary lineage by transcription factor manipulation provides a paradigm for instructing differentiation of ES cells for biopharmaceutical screening and cell therapy applications.

Animals↗

PubMed2004

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons.

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.

Animals↗

PubMed2003

Expression profiling using cDNA microarrays.

Microarray technology has become increasingly useful in measuring expression levels of a large number of genes and part of a repertoire of functional genomic tools. We describe the methods of cDNA microarray preparation, the use, data collection, and initial data processing. The cDNA fragments are first prepared by polymerase chain reaction (PCR), and then attached to a solid substrate, such as a chemically treated glass slide. Robotic machines spot the prepared cloned cDNA samples in a miniaturized gridded pattern, so that nanoliter amounts of tens of thousands cDNA samples are bound to a single 7.5 x 2.5 cm glass slide. Probes are generated from RNA samples of test and control tissues by incorporating Cyanine dyes (Cy3 or Cy5) in reverse-transcribed products. Probes from a test sample are labeled with one of two Cy dyes and mixed in equal amounts with probes from a control sample labeled with the second dye. The glass slides containing the cDNA microarray are hybridized with the mixed Cy-labeled probes, washed, dried, and scanned using laser scanners with an optimized wavelength to excite each Cy dye. The emission image patterns for each dye are captured by a digital camera using micro-optics and processed into numerical values that positively correlate with quantitative levels of mRNA for each cDNA spot on the slide. The collected data is then further processed, normalized across experiments, and examined via numerous statistical and mathematical approaches to infer changes in expression levels of particular genes due to the treatment tested.

DNA, Complementary↗

PubMed2002

Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.

In the developing mouse embryo the first definitive (transplantable-into-the-adult) haematopoietic stem cells/long-term repopulating units (HSC/RUs) emerge in the AGM region and umbilical vessels on 10-11 days post coitum (d.p.c.). Here, by limiting dilution analysis, we anatomically map the development of definitive HSC/RUs in different embryonic tissues during early colonisation of the liver. We show that by day 12 p.c. the mouse embryo contains about 66 definitive HSC/RUs (53 in the liver, 13 in other tissues), whereas on the previous day the total number of definitive HSC/RUs in the entire conceptus is only about 3. Owing to the length of the cell cycle this dramatic increase in the number of definitive HSC/RUs in only 24 hours is unlikely to be explained purely by cell division. Therefore, extensive maturation of pre-definitive HSCs to a state when they become definitive must take place in the day 11-12 embryo. Here we firstly identify the numbers of HSCs in various organs at 11-13 d.p.c. and secondly, using an organ culture approach, we quantitatively assess the potential of the aorta-gonadmesonephros (AGM) region and the yolk sac to produce/expand definitive HSC/RUs during days 11-12 of embryogenesis. We show that the capacity of the AGM region to generate definitive HSC/RUs is high on 11 d.p.c. but significantly reduced by 12 d.p.c. Conversely, at 12 d.p.c. the YS acquires the capacity to expand and/or generate definitive HSCs/RUs, whereas it is unable to do so on 11 d.p.c. Thus, the final steps in development of definitive HSC/RUs may occur not only within the AGM region, as was previously thought, but also in the yolk sac microenvironment. Our estimates indicate that the cumulative activity of the AGM region and the yolk sac is sufficient to provide the day 12 liver with a large number of definitive HSC/RUs, suggesting that the large pool of definitive HSC/RUs in day 12 foetal liver is formed predominantly by recruiting 'ready-to-use' definitive HSC/RUs from extra-hepatic sources. In accordance with this we observe growing numbers of definitive HSC/RUs in the circulation during days 11-13 of gestation, suggesting a route via which these HSCs migrate.

Animals↗