Lineage tracing and fate mapping 1
BSE652 Lineage tracingandfate mapping 1 Lecture 2 7th Jan 2016 OR OR What is lineage tracing Schematic of a late embryo/adult
Schematic of an early embryo Schematic of an early embryo Schematic of an early embryo Schematic of a late embryo/adult What is a Fate map? This chart or graphical representation detailing the fate of each part of an early embryo in the adult animal is referred to as a fate map At a single cell level fate mapping is known as lineage tracing
Why is it important to learn the lineage of a cell or fate map of an early embryo? To know the ground truth To understand the journey, change in molecular signature and microenvironment, of a differentiated cell To affect specific molecular manipulations in specific cell populations Methods of lineage tracing
Direct observation Methods of lineage tracing
Conklin (1905) - Tunicates Methods of lineage tracing
Direct observation Conklin (1905) - Tunicates Direct observation, time lapse video microscopy followed by cell ablation Limitations: This method currently gives the best resolution in space and time, but has the disadvantage that the number of cells which can be followed in a single individual is limited by the short-term memory of the observer. Cell number and opaque embryo DYE LABELING Schematic showing agar chips with vital dyes applied onto the surface of an early stage amphibian embryo (top). These dyes label regions within later stage embryos (bottom) (based on Vogt, 1929; adapted from Gilbert, 2000). Use of lipophilic dye conjugated with large molecules (dextran) Taking advantage of cell proliferation properties EdU First observation made in chicken, Followed up in mice EdU , Day10 EdU pulse chase to lineage trace proliferative cells
EdU injected intraperitoneally with 100 mg/kg body weight into 13.5/15.5 dpc pregnant females EdU 2 Hour So, we wanted to lineage trace the proliferative population of cells. For this purpose we injected EdU in pregnant females with to label proliferating cells in the skeletal tissues. In the first set, In this experiment, two sets of mice were taken; in the first set around EdU was injected and chased for 2 hours. In the second set EdU injected was chased for 24 hours. The data are as follows. EdU pulse chase to lineage trace proliferative cells
So, we wanted to lineage trace the proliferative population of cells. For this purpose we injected EdU in pregnant females with to label proliferating cells in the skeletal tissues. In the first set, In this experiment, two sets of mice were taken; in the first set around EdU was injected and chased for 2 hours. In the second set EdU injected was chased for 24 hours. The data are as follows. EdU 2 hour pulse EdU pulse chase to lineage trace proliferative cells
EdU injected intraperitoneally with 100 mg/kg body weight into 13.5/15.5 dpc pregnant females EdU 2 Hour So, we wanted to lineage trace the proliferative population of cells. For this purpose we injected EdU in pregnant females with to label proliferating cells in the skeletal tissues. In the first set, In this experiment, two sets of mice were taken; in the first set around EdU was injected and chased for 2 hours. In the second set EdU injected was chased for 24 hours. The data are as follows. EdU 24 Hour Distal Proliferative Zone (DPZ) cells indeed contribute to the growth of interzone
EdU 24 hour pulse chase EdU 2 hour pulse Slowly proliferating interzone cells do not account for the EdU incorporation after 24 hours DPZ cells contribute to articular cartilage Retinal development and differentiation Retroviral infection The neural retina has seven different cell types: six neuronal and one glial
These cells are organized into three nuclear layers: GCL (Ganglion cell layer), containing the cell bodies of the ganglion cells. INL (Inner nuclear layer) containing the cell bodies of the bipolar cells, horizontal cells and amacrine cells. ONL (outer nuclear layer), containing the nuclei of the photoreceptors e.g. Rods and cones. In addition to these nuclear layers there are two plexiform layers: IPL (inner plexiform layer), containing the synapses and processes of the bipolar, amacrine and ganglion cells. OPL (inner plexiform layer), containing the synapses and processes of the photoreceptor, bipolar and horizontalcells. The cell types are produced in an orderly manner which is generally conserved among vertebrates.
Ganglion cells are produced first. Followed closely by cone, horizontal and amacrine cells Then come rod and bipolar cells The cell type to be born are the Muller glia cells. During retinogenesis these seven cell types derive from a common population of retinal progenitor. Lineage analyses has revealed that retinal progenitors are multipotent and retain their ability to generate different cell types up to the final cell division. If an individual retinal progenitor is injected with a genetic marker, that marker will be seen in a strip that includes all different cell types in the retina. Determination of the lineage of a neuroblast in the rat retina. (A) Technique whereby a virus containing a functional -galactosidase gene is injected into the back of the eye of a newborn rat to infect some of the retinal precursor cells. After a month to 6 weeks, the eye is removed and the retina is stained for the presence of -galactosidase. (B) Stained cells forming a strip across the neural retina, including five rods (r), a bipolar neuron (bp), a rod terminal (t), and a Mller glial cell (mg). To distinguish between the possibilities of (1) the same progenitor cell gives rise to two different daughters Vs (2) two closely spaced DISTINCT cells give rise to distinct daughters This study was made even more comprehensive by co-relating with cell division Another method of permanent labelling of cells: Quail-Chick Chimeras
Methods in Molecular Biology Volume 97, 1999, pp Quail-Chick Chimeras Marie-Aime Teillet, Catherine Ziller, Nicole M. Le Douarin