Feat. an article: In Utero Fate Mapping reveals distinct migratory pathways and fates of neurons born in Mammalian Basal Forebrain by Hynek Wichterle...
Feat. an article: In Utero Fate Mapping reveals distinct migratory pathways and fates of neurons born in Mammalian Basal Forebrain by Hynek Wichterle...
Feat. an article: In Utero Fate Mapping reveals distinct migratory pathways and fates of neurons born in Mammalian Basal Forebrain by Hynek Wichterle...
Feat. an article: In Utero Fate Mapping reveals distinct migratory pathways and fates of neurons born in Mammalian Basal Forebrain by Hynek Wichterle...
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Objectives:
To understand what is Fate Mapping and
its history. To understand the cell fate, the commitment and the mechanism of developmental commitment involved. To know the different techniques used in Fate Mapping and its different processes.
History & Proponents: 1880 The first fate maps were made. 1905 The first comprehensive collection of Ascidian (sea squirt) fate maps was published by Edwin Conklin. 1929 Walter Vogts research, the amphibian blastula, divides into three regions: animal, marginal, and vegetal. Each of these areas houses progenitors of the cells that will make up the future organs of the organism.
1969 Nicole Le Douarin pioneered the use of chickquail chimaeras for fate mapping. 1971 More detailed fate maps have been created for the frog Xenopus, such as the one published by Osamu Nakamura and Keiko Kishiyama. History & Proponents 1974 Several experiments were initiated by Sydney Brenner, a biologist and 2002 Nobel Prize winner in Physiology or Medicine. These he chose the nematode worm for study because of its rapid period of embryogenesis and very few cell types. Balinskys An Introduction to Embryology (1981) showed an image of the fate map of the amphibians Discoglossus and Ambystoma similar to those created by Vogt for Xenopus. History & Proponents More recent illustrations, in Hake and Wilts Principles of Developmental Biology (2004) showed how a fate map can be made using an amphibian egg. Scott Gilberts Developmental Biology (2006) showed fate maps for several different model organisms, including the zebra fish, frog, mouse, and chick embryos.
History & Proponents Cell Fate & Commitment Developmental Hierarchy The number of different cell types in the embryo increases as development proceeds, but new cell types arise from particular pre- existing cell types through hierarchical series of decisions. Cell Fate & Commitment Cell Fate and Potency Cell fate describes the range of cell types a particular cell can give rise to during normal development, while cell potency describes the range of cell types that a cell can give rise to in all possible environments. Cell Fate & Commitment Fate Maps A fate map is a map of an embryo showing which parts of the embryo give rise to a particular adult tissues. Fate maps are generated by marking or labeling cells in the early embryo and following their progress through development. Cell Fate & Commitment Levels of Developmental Commitment In animals, cells become committed to certain fates in stages, first reversibly and then irreversibly. A nave cell may receive information in the form of cytoplasmic determinants or inductive signals that specifies its fate, but this fate can be altered by placing the cell in a different environment. A cell becomes determined when its fate is irreversible. This coincides with the loss of competence to follow alternative developmental pathways. Mechanisms of Developmental Commitment How Cells Differentiate? To differentiate, cells must begin to synthesize new proteins. This requires the selective expression or activation of regulatory molecules such as transcription factors that control which proteins are synthesized in the cell. Mechanisms of Developmental Commitment Cytoplasmic Determinants Are molecules in the cytoplasm of a cell that help to determine cell fate. Induction Is a process whereby one cell or group of cells can influence the developmental fate of another, and is a common strategy to control differentiation and pattern formation in development. Mechanisms of Developmental Commitment COMPETENCE Induction relies both on ability of the including cell to produce the signal and the ability of the responding cell to receive the signal and react in the appropriate manner. Mechanisms of Developmental Commitment Instructive and Permissive Induction Instructive induction occurs when the responding cell has a choice of fates, and the inductive signal instructs the cell to adopt one of those fates in preference to the other. Permissive induction occurs when the responding cell is already committed to a certain developmental pathway, but needs the inductive signal to continue towards differentiation. Mechanisms of Developmental Commitment Lateral Inhibition and the Community Effect In lateral inhibition, differentiated cells arise in a regularly spaced pattern within a group of equivalent undifferentiated cells due to random fluctuations in levels of a ubiquitous signal that inhibits differentiation.
Mechanisms of Developmental Commitment In the community effect, populations of cells can change their collective fate by secreting enough of an inductive signal to reach a critical concentration threshold, whereas isolated cells follow a different developmental pathway because they cannot produce enough of the signal on their own. Different Fate Mapping Techniques In 1905 Edwin G. Conklin conducted the first cell lineage experiments, which involved following the progenitor cells of the embryo of the tunicate Styela partita.
In 1929 Walter Vogt invented a process in which vital dye and agar chips are used to stain a specific region of a developing amphibian embryo. To do this, Vogt spread dye and agar on a microscope plate and allowed it to dry. He then cut small pieces of the dried agar and applied it to a desired part of the embryo. Radioactive labeling and fluorescent dyes are both relatively simple experimental tools that use a donor and a host embryo to follow cell migration. The donor embryo is treated with dye or irradiated and a graft from the donor is removed and placed onto the host embryo where it joins the developmental process. Another process for fate mapping was invented by Nicole Le Douarin, a developmental biologist who created chimeras, or animals with two or more sets of genetically distinct cells. Le Douarin removed a portion of neural tube and neural crest from a chick embryo and replaced it with an identical portion of neural tube and neural crest from a quail embryo at the same stage of development. Le Douarin also discovered that Feulgen stain distinguishes quail cells from chick cells, which allowed her to trace the migration of quail cells. Genetic fate mapping (GFM) uses two genetically engineered alleles, one of which expresses a site-specific recombinase, such as cyclization (Cre) or flipase (Flp), while the other contains a reporter allele such as green fluorescent protein (GFP). Utero fate mapping A cell is grafted from a donor animals and dyed and was injected into the embryo inside the uterus.
Inducible fate mapping (GIFM). This technique generates the Cre fusion proteins used in GFM with a tamoxifen-responsive estrogen receptor ligand binding domain (CreER). CreER is removed from the cytoplasm of the cell via heat shock protein 90 (Hsp90). Introduction Cell migration is an important determinant of brain structure. Most, if not all, nerve cells have to migrate from the sites where they are born to places where they terminally differentiate and integrate into the brain circuitry. Radial migration of young neurons from germinal regions lining lateral ventricles to more superficial layers of the neocortex has been recognized as a prerequisite for proper morphogenesis and function of the cerebral cortex (Caviness and Rakic, 1978; Sidman and Rakic, 1973; Walsh and Goffinet, 2000). Introduction Radial migration could explain the radial organization of the cerebral cortex and the inside-out sequence of cortical formation, and as such it has been for many years considered to be the principal mode of cell translocation in the developing cortex (Rakic, 1988). This view began to change when retroviral lineage tracings revealed that a large number of neurons do not follow the radial path, but disperse tangentially throughout the developing neocortex (Price and Thurlow, 1988; ORourke, 1992; Walsh and Cepko, 1992). It has been proposed that tangentially and radially migrating neurons belong to different cell pools that segregate early in the embryonic development (Tan and Breen, 1993; Tan et al., 1998). Finally, several experiments suggested that many of the tangentially migrating neurons are not born in the cortical neuroepithelium as was always assumed, but instead originate outside the neocortex in the basal forebrain in regions called ganglionic eminences (reviewed by Anderson et al., 1999; Parnavelas, 2000). Introduction From these findings emerged a new concept of cortical development, which proposes that two separate populations of neurons participate in corticogenesis. One population consists of the radially migrating neurons, which are born in neocortical ventricular zone and probably give rise to the principal pyramidal neurons of the neocortex. The second population includes neurons born in ganglionic eminences that migrate tangentially into the neocortex and probably differentiate into the cortical non- pyramidal neurons (Anderson et al., 1999; Parnavelas, 2000).
Introduction Materials and Methods Donor cell preparation Ventricular and subventricular zones from ganglionic eminences were dissected from 5-15 E13.5 mouse embryos as described previously (Wichterle et al., 1999). Swiss-Webster (Taconic), CD-1 (Charles River) or transgenic mice expressing human placental alkaline phosphatase in all cells (DePrimo et al., 1996) were used as donor animals.
Materials and Methods Transplantation in utero High density cell suspension (~800,000 cells/ml) was front-loaded into beveled glass micropipettes (~50 mm diameter) that were prefilled with mineral oil and mounted on a microinjector (modified version of Narishige). Cells were allowed to settle inside the pipette and the excess cell-free medium was expelled. The recipient pregnant mice (E13.5) were anesthetized, uterine horns were exposed through an intraperitoneal incision and animals were mounted under the biomicroscope as described previously (Liu et al., 1998; Olsson et al., 1997). The tip of the micropipette was inserted into the LGE or MGE under real-time ultrasound guidance and 10-20 nl of cell suspension was injected. Analysis of cell migration Transplanted embryos (~60% of injected embryos survived) were collected at 1, 2 and 4 days after transplantation or as 3-week and 4-month-old animals. Three to six successfully transplanted animals were analyzed for each time-point and region (~50% of surviving injected animals contained grafted cells). Materials and Methods Fig. 1. Distribution of MGE cells one (E14.5, A-C), two (E15.5, D-F) and four (E17.5, G-I) days after homotopic transplantation. Coronal sections of embryonic mouse brains containing MGE cells labeled with PKH26 fluorescent dye before transplantation. Graft- derived cells are black or dark blue.
Fig. 2. MGE cells in the subventricular and marginal zones at two (E15.5) and four (E17.5) days after transplantation. Fig. 3. MGE cells expressing alkaline phosphatase were grafted into the wildtype E13.5 MGE. Fig. 4. Morphology of MGE-derived neurons stained for alkaline phosphatase. Fig. 5. Immunohistochemical characterization of grafted MGE (A-C) and LGE (D-F) cells in the neocortex (NCx; A,B,F) and striatum (Str; C,D,E). Fig. 6. Homotopic transplants of PKH26-labeled LGE cells. Fig. 7. Alkaline phosphatase- expressing LGE cells in 3-week- old animals. (B,D,E) Two to three fused photographs taken in different focal planes. Discussion Summary Recent studies suggest that neurons born in the developing basal forebrain migrate long distances perpendicularly to radial glia and that many of these cells reach the developing neocortex. This form of tangential migration, however, has not been demonstrated in vivo, and the sites of origin, pathways of migration and final destinations of these neurons in the postnatal brain are not fully understood Summary Using ultrasound-guided transplantation in utero, we have mapped the migratory pathways and fates of cells born in the lateral and medial ganglionic eminences (LGE and MGE) in 13.5-day-old mouse embryos. We demonstrate that LGE and MGE cells migrate along different routes to populate distinct regions in the developing brain... We show that LGE cells migrate ventrally and anteriorly, and give rise to the projecting medium spiny neurons in the striatum, nucleus accumbens and olfactory tubercle, and to granule and periglomerular cells in the olfactory bulb. By contrast, we show that the MGE is a major source of neurons migrating dorsally and invading the developing neocortex Summary MGE cells migrate into the neocortex via the neocortical subventricular zone and differentiate into the transient subpial granule neurons in the marginal zone and into a stable population of GABA-, parvalbumin- or somatostatin- expressing interneurons throughout the cortical plate. Summary END***