Fate Maps PDF

FATE MAPS  FATE MAPS Fate map is a diagrammatic representation of the prospective fate of each part of an embryo at an early stage of development Embryonic regions with a distinct fate are called Primordia / Rudiments Fate maps change over time –as- cells multiply &...

FATE MAPS  FATE MAPS Fate map is a diagrammatic representation of the prospective fate of each part of an embryo at an early stage of development Embryonic regions with a distinct fate are called Primordia / Rudiments Fate maps change over time –as- cells multiply & move relative to each other A series of fate maps at consecutive stages shows the progression of different cells or regions through longer periods of development Significance: – Essential tools in most embryological experiments – Help to trace cell lineage by following the fate of parts of early embryo during development – Helps understand the mechanism of morphogenetic movements during gastrulation A chart showing the fate of each part of an early embryo, in a particular blastula stage is called fate maps. It is done because the correct interpretation of gastrulation is impossible without the knowledge of the position which are the presumptive germinal layers (Ectoderm, Mesoderm and Endoderm) occupy in blastula. Fate mapping is a method used in developmental biology to study the embryonic origin of various adult tissues and structures. The "fate" of each cell or group of cells is mapped onto the embryo, showing which parts of the embryo will develop into which tissue. When carried out at single-cell resolution, this process is called cell lineage tracing. The fate of the cells is determined in the blastula or early gastrula, so fate mapping can be done at these stage. FATE MAPS OF VERTEBRATES AT THE EARLY GASTRULA STAGE: Despite the different appearances of the adult animals, fate maps of these four vertebrates show numerous similarities among the embryos. The cells that will form the notochord occupy a central dorsal position, while the precursors of the neural system lie immediately anterior to it. The neural ectoderm is surrounded by less dorsal ectoderm, which will form the epidermis of the skin. All are dorsal surface views: A- indicates the anterior end of the embryo, P- the posterior end. FATE MAPPING TECHNIQUES Natural marking Artificial marking Genetic marking NATURAL MARKERS Cytoplasm of fertilized egg has natural colour differences in it’s various regions. Ex: fertilized eggs of Ascidians e.g. Ciona, Tunicate (Sea squirt) Styela partita (Cynthia) Upper A light Protoplasm presumptive epidermal hemisphere ectoderm Postero- A yellow crescent presumptive mesoderm ventrally Antero-dorsally A Grey crescent Presumptive neural ectoderm and notochord Lower A dark grey yellow presumptive endoderm vegetative area substance On the basis of pigmentation four differentiated areas maybe distinguished in the egg cytoplasm of Styela partita.  ARTIFICIAL MARKING 1.Vital staining technique 2. Carbon particle marking technique 3.Radioactive marker technique ARTIFICIAL METHODS VITAL DYES: Vogt (1925) – Vital dyes stain cells but do not kill them: Nile Blue Sulphate, Neutral red, Bismark brown, Janus green – spread dye mixed with agar / cellophane on the slide  dried  pressed against a chosen area of blastula for a short period  stain diffuses to blastomeres  stained cells’ movements are followed within the embryo – Several areas can be marked separately simultaneously & their fate traced – But become diluted with each cell division Advantages- Stain retained for considerable time. Doesn’t interfere with cell processes. Only those cells receiving the dye retain dye; doesn’t diffuse into the neighbouring cells. Vital staining technique Use-Vital stains like Nile blue sulphate , Neutral red , Janus green , Bismark brown. Stain Pressed against Stain carrier(celloph the chosen area diffuses ane/agar) on blastula into cell  CARBON PARTICLES: – N. Spratt (1946) – fate map of chick: C-particles stick on the cell surface & used as markers to trace the cell fate – William W. Ballard (1981) – modified technique: injected C- particles or chalk particles inside the particular region of teleost embryo for tracing its fate – But become diluted with each cell division RADIOACTIVE MARKERS: C14, P32, H3 in Chick embryo – Cells with these markers are studied by autoradiography to trace their fate – Tritiated thymidine labels the nuclei when incorporated into the DNA of cells – A region of interest is cut from host embryo  replaced by radioactive graft from donor embryo – Limitation: become diluted with each cell division; exposure to radioactivity HISTOCHEMICAL STAINS: Enzyme specific staining of embryonic cells  can be visualized by adding appropriate substrate for its enzy,atic activity – e.g. enz. Horseradish peroxidase (HRP) Radioactive labelling methods Use-Radioactive markers like Tritiated thymidine Cells of Embryo 1 labelled with radioactive metabolite. A part of Embryo 1 is excised and grafted to normal Embryo 2 Autoradiography  FLUORESCENT DYES: – fluorescent dyes conjugated to large, metabolically inert carrier molecules, that will not cross cell membranes or pass through gap junctions  microinjected into one or more cell, all descendants of the injected cell are labeled distinctly under a fluorescent microscope – e.g. fluorescently labeled DEXTRAN: Fluorescein Dextran Amine (FDA) & Rhodamine Dextran Amine (RDA) – e.g. fluorescent carbocyanine dyes: dialkyl indocarbocyanine (DiI) & dialkyl oxocarbocyanine (DiO) are lipophilic membrane stains that diffuse in the cell  produce red & green emissions resp.  GENETIC MARKERS – One way of permanently marking cells and following their fates is to create embryos in which the same organism contains cells with different genetic constitutions. – Advantages: donot spread to neighboring cells, If stably expressed, are inherited by the descendants of the marked cell – Limitations: Low efficiency of introducing the gene in the cell – e.g. used to create Chimeric Embryos: by Xenoplastic transplantation of embryonic grafts from animal of interest having different genetic constitution but similar development pattern e.g. Chick-quail chimeras are made by grafting embryonic quail cells inside a chick embryo while the chick is still in the egg. Chicks and quail embryos develop in a similar manner (especially during the early stages), and the grafted quail cells become integrated into the chick embryo and participate in the construction of the various organs The chick that hatches will have quail cells in particular sites, depending on where the graft was placed. By seeing where these cells migrate, fine-structure maps of the chick brain and skeletal system have been produced  Transgenic DNA chimeras most animals - difficult to make a chimera from two species. Hence, better to transplant cells from a genetically modified organism (retrovirus)  the genetic modification can then be traced only to those cells that express it. Retrovirus marking: incorporating retrovirus engineered reporter gene into DNA of host cells  expression of reporter gene  histochemical/fluorescent marking of gene products Expression of host DNA to express Green Fluorescent Protein (GFP) - naturally occuring in some jellyfishes – When the retrovirus infected embryonic cells are transplanted into a wild-type host, only the donor cells will express GFP BRAINBOW: a recent technique used to tag neurons in brain – fluorescent proteins are used to tag different proteins in the cell  an array of colours are generated by different expression of distinct fluorescent proteins  Experiment: Fate mapping with transgenic DNA showed that the neural crest is critical in making the gut neurons Freem et al (2012) used transgenic techniques to study the migration of neural crest cells to the gut of chick embryos, where they form the neurons that coordinate peristalsis—the muscular contractions of the gut necessary to eliminate solid waste. The parents of the GFP-labeled chick embryo were infected with a replication-deficient virus that carried an active gene for GFP This virus was inherited by the chick embryo and expressed in every cell  glowed green when placed under ultraviolet light They then transplanted the neural tube and neural crest of a GFP- transgenic embryo into a similar region of a normal chick embryo A day later, they could see GFP-labeled cells migrating into the stomach region and by 7 days, the entire gut showed GFP staining up to the anterior region of the hindgut