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Objectives:

Objectives: • To understand what is “Fate Mapping” and its history. • To understand the cell

• 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 Vogt’s 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.

History & Proponents…

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.

• Balinsky’s 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… • 1974 – Several experiments were initiated by Sydney Brenner, a biologist and

History & Proponents…

More recent illustrations, in Hake and Wilt’s Principles of Developmental Biology (2004) showed how a fate map can be made using an amphibian egg.

• Scott Gilbert’s Developmental Biology (2006) showed fate maps for several different model organisms, including the

zebra fish, frog, mouse,

and chick embryos.

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 Developmental Hierarchy  The number of different cell types in the embryo
Cell Fate & Commitment Developmental Hierarchy  The number of different cell types in the embryo

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… Cell Fate and Potency  Cell fate describes the range of cell
Cell Fate & Commitment… Cell Fate and Potency  Cell fate describes the range of cell

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… Fate Maps  A fate map is a map of an embryo
Cell Fate & Commitment… Fate Maps  A fate map is a map of an embryo

Cell Fate & Commitment…

Levels of Developmental Commitment In animals, cells become committed to

certain fates in stages, first reversibly and then irreversibly. A naïve 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.

Cell Fate & Commitment… Levels of Developmental Commitment  In animals, cells become committed to certain
Cell Fate & Commitment… Levels of Developmental Commitment  In animals, cells become committed to certain

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 How Cells Differentiate?  To differentiate, cells must begin to synthesize new
Mechanisms of Developmental Commitment How Cells Differentiate?  To differentiate, cells must begin to synthesize new

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… Cytoplasmic Determinants  Are molecules in the cytoplasm of a cell that
Mechanisms of Developmental Commitment… Cytoplasmic Determinants  Are molecules in the cytoplasm of a cell that

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… COMPETENCE  Induction relies both on ability of the including cell to
Mechanisms of Developmental Commitment… COMPETENCE  Induction relies both on ability of the including cell to

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… Instructive and Permissive Induction  Instructive induction occurs when the responding cell
Mechanisms of Developmental Commitment… Instructive and Permissive Induction  Instructive induction occurs when the responding cell

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… L ateral Inhibition and the Community Effect  In lateral inhibition, differentiated
Mechanisms of Developmental Commitment… L ateral Inhibition and the Community Effect  In lateral inhibition, differentiated

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.

Mechanisms of Developmental Commitment…  In the community effect, populations of cells can change their collective
Mechanisms of Developmental Commitment…  In the community effect, populations of cells can change their collective

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.

Different Fate Mapping Techniques  In 1905 Edwin G. Conklin conducted the first cell lineage experiments,
Different Fate Mapping Techniques  In 1905 Edwin G. Conklin conducted the first cell lineage experiments,

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.

 In 1929 Walter Vogt invented a process in which vital dye and agar chips are
 In 1929 Walter Vogt invented a process in which vital dye and agar chips are

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.

 Radioactive labeling and fluorescent dyes are both relatively simple experimental tools that use a donor
 Radioactive labeling and fluorescent dyes are both relatively simple experimental tools that use a donor
  • 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.

 Another process for fate mapping was invented by Nicole Le Douarin, a developmental biologist who
 Another process for fate mapping was invented by Nicole Le Douarin, a developmental biologist who

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).

 Genetic fate mapping (GFM) uses two genetically engineered alleles, one of which expresses a site-specificgreen fluorescent protein (GFP). " id="pdf-obj-20-18" src="pdf-obj-20-18.jpg">
 Genetic fate mapping (GFM) uses two genetically engineered alleles, one of which expresses a site-specificgreen fluorescent protein (GFP). " id="pdf-obj-20-20" src="pdf-obj-20-20.jpg">
 Genetic fate mapping (GFM) uses two genetically engineered alleles, one of which expresses a site-specificgreen fluorescent protein (GFP). " id="pdf-obj-20-22" src="pdf-obj-20-22.jpg">
  • Utero fate mapping

A

cell

is

grafted from a

donor animals and dyed and

was

injected

into

the

inside the uterus.

embryo

 Utero fate mapping A cell is grafted from a donor animals and dyed and was
 Utero fate mapping A cell is grafted from a donor animals and dyed and was

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).

 Inducible fate mapping (GIFM). This technique generates the Cre fusion proteins used in GFM with
 Inducible fate mapping (GIFM). This technique generates the Cre fusion proteins used in GFM with
Introduction
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; O’Rourke, 1992; Walsh and Cepko, 1992).

Introduction…

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).

Materials and Methods
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

Swiss-Webster

(Taconic),

(Wichterle

et

al.,

1999).

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.

Materials and Methods…

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).

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…
Discussion…
Summary
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...

Summary…

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.

Thank You!!!

Thank You!!!

Thank You!!!

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