Download Part 1 - Instructure

Part 1:
Cell Fate Determination
pg. 1
Abridged and modified (by A. Schivell) from:
Carlson (1996) Foundations of Embryology, 6th ed, Ch. 1.
Restriction and Determination
Within the zygote lies the capability to form an entire organism. In many vertebrates
the individual cells resulting from the first few divisions after fertilization retain this capability.
The embryological term for these cells is totipotent. As development continues, most cells
gradually lose the ability to form all the types of cells that are found in the adult body. It is as
if they were funneled into progressively narrower channels. The reduction of the
developmental options permitted to a cell is called cell fate restriction. Little is known about
the mechanisms that bring about restriction, and the sequence and time course of restriction
vary considerably from one species to another. An example representing a general pattern of
restriction during development may serve to clarify the concept (Fig. 1-20).
pg. 2
Shortly after fertilization the zygote undergoes a series of cell divisions, called cleavage.
Early in cleavage the cells commonly remain totipotent. The period of cleavage comes to an
end when certain cells in the embryo undertake extensive migrations and rearrange
themselves into three primary germ layers during a process known as gastrulation. Named
on the basis of their relative positions, the outermost layer is the ectoderm, the innermost is
the endoderm, and between the two is the mesoderm. By this time at least one stage of
restriction has usually occurred, so that the cells of the three germ layers are now locked into
separate developmental channels and are no longer freely interchangeable – they are known
as pluripotent. For example, the potential options open to the cells of the ectoderm are many,
such as epidermis (skin) and nervous tissue, but not muscle or blood. In the next major
developmental event, part of the ectoderm becomes thickened and is henceforth committed to
forming the brain, the spinal cord, and other associated structures. This stage of development
is commonly called neurulation. The remainder of the ectodermal cells can no longer form
these structures and have thus undergone another phase of restriction. Soon, as a result of
tissue interactions with the newly forming brain, groups of ectodermal cells become
committed to forming the lens and inner ear, whereas the remainder of the ectoderm
ultimately loses this capacity.
Subsequent developmental events see the ectoderm further subdivided into groups of
cells destined to form cornea; hair, scales, or feathers; cutaneous glands; or simply epidermis.
When restriction has proceeded to the point at which a group of cells becomes committed to a
single developmental fate (e.g., the formation of cornea), we say that determination of these
cells has taken place. Thus, determination represents the final step in the process of restriction.
The mechanisms that bring about determination of various groups of cells are receiving
intensive study, but much remains to be learned. Often, tissue interactions called inductions
shortly precede the process of determination (and some phases of restriction) and are almost
certainly involved in some manner.
Induction
One of the most remarkable features of embryonic development is the precision with
which developmental signals are generated, transmitted, and received. These signals are
generally proteins that can interact with receptors or proteins in another cell. One of the most
important varieties of embryonic signaling is the process of induction. By induction we mean
an effect of one embryonic tissue (the inductor) on another, so that the developmental course
of the responding tissue is qualitatively changed from what it would have been in the absence
of the inductor. A classic example of embryonic induction is the formation of the lens of the
eye as a result of the inductive action of the optic cup on the overlying ectoderm.
Inductive interactions occur throughout much of embryonic development and even
into postnatal life. In vertebrates, the first major inductive event involves the induction of
mesoderm in the cleaving embryo. This is followed by the induction of the nervous system
shortly after gastrulation. The nervous system itself then induces other structures (often
sensory organs). These tissues sometimes induce the formation of other structures through
pg. 3
tertiary inductions. The formation of almost all internal organs occurs through inductive
interactions.
Differentiation
Whereas restriction and determination signify the progressive limitation of the
developmental capacities of cells in the embryo, differentiation refers to the actual
morphological or functional expression of the portion of the genome that remains available to
a particular cell or group of cells. Differentiation is really the process by which a cell becomes
specialized, and the final product is called a differentiated cell. Although in many respects
differentiation is a cellular event, a cell rarely undergoes differentiation in isolation. Typically,
differentiation in vivo is a communal process that occurs within groups of similar cells.
There are three different ways of looking at differentiation:
1. From the biochemical standpoint, differentiation may be viewed as the process by
which a cell chooses one or a few specialized gene expression pathways, for example, the
synthesis of hemoglobin by erythrocytes or of specific crystalline proteins by the lens.
2. Functional differentiation can be looked upon as the development of contractility by
muscle fibers or as the development of conductivity along a nerve.
3. From the morphological standpoint, final differentiation is represented by a myriad
of specific cell shapes and structures.
pg. 4
Part 2:
Cleavage and Formation of the Blastula in
Birds
pg. 1
Abridged/modified (by A. Schivell) from: Carlson (1996) Foundations of Embryology,
6th ed.
Cleavage and Formation of the Blastula in Birds
A newly fertilized egg, which is about to undergo cleavage, contains a whitish
germinal disk about 3 mm in diameter (a chick egg overall is about 30 mm diam.) The
first cleavage furrow begins to appear near the center of the germinal disk during late
anaphase of the first mitotic division after fertilization. The cleavage furrow lies in the
plane of the metaphase plate during metaphase, and microfilaments are found at the
base of the cleavage furrow. The sequence of avian cleavage is not always regular, and
after about the third cleavage division, it is not synchronous. Nevertheless, the mitotic
spindles align themselves so that the subsequent cleavage furrow forms at right angles
to the preceding one (Fig. 5-14). The fourth cleavage furrow is a circumferential one,
which cuts a central row from a peripheral row of blastomeres (new cells).
pg. 2
The blastomeres formed by the first few cleavage divisions are unusual in having
their tops and sides bounded by plasma membranes but their basal surfaces open to the
underlying yolk. Further cleavage in the early disk of embryonic cells, now called a
blastoderm, results in the radial extension of the embryo.
In addition to surface cleavages, the 32-cell embryo shows cleavage planes of an
entirely different character. These cleavages appear below the surface and parallel to it.
They establish a superficial layer of nucleated cells that are completely delimited by
plasma membranes. These superficial cells rest on a layer of cells that on their deep
faces are continuous with the yolk. Continuous divisions of the same type eventually
establish several strata of superficial cells. The divisions progress centrifugally as the
blastoderm (cellular tissue) increases in size but does not extend to its extreme margin.
The peripheral margin remains a single cell in thickness, and the cells there lie
unseparated from the yolk. By the time the embryo contains about 100 cells, the
blastoderm is underlain by a subgerminal cavity (Fig. 5-10c).
C
D
Figure 5-10: C) Diagram showing the subgerminal space in a chick embryo. D)
Diagram of the chick “blastodisk” in a chick embryo.
pg. 3
During the first few days of development, the blastoderm expands over the
surface of the yolk. Although partly related to the increasing number of cells in the
blastoderm, blastodermal expansion also involves an active migratory process. Cells at
its edge are attached to the overlying vitelline membrane (sheet of glycoproteins
surrounding the egg), and they use the vitelline membrane as a substrate for their
migration.
After a number of cleavage cycles, the shedding of individual cells begins from
the undersurface of the area of the blastoderm that is farthest away from the source of
gravity. The area from which the cells are first shed becomes fixed as the posterior end
of the embryo. The cell shedding spreads toward the future anterior end of the
embryo. The central portion of the blastoderm, thinned out by the shedding of cells
and underlain by the subgerminal cavity, is called the area pellucida. Surrounding the
area pellucida is the area opaca, a region where the cells of the blastoderm still abut
directly onto the yolk.
At about the time the egg is laid, individual cells or aggregates of cells shed from
the lower surface of the blastoderm coalesce to form a thin disklike layer called the
primary hypoblast (Fig. 5-10d). This process occurs first, and to a greater extent, at the
posterior end of the embryo. The primary hypoblast is separated from the outer layer
of the blastoderm, called the epiblast, by a thin cavity, the blastocoel. The primary
hypoblast, which ultimately forms extraembryonic endoderm, possesses an inherent
polarity, which it confers on the embryo proper, which is represented at this stage by
the early epiblast. The polarity and location of the primary hypoblast determine the
location and direction of the future primitive streak by a form of inductive interaction.
pg. 4
Part 3:
Gastrulation in Birds
pg. 1
Abridged/modified (by A. Schivell) from: Carlson (1996) Foundations of Embryology, 6th ed,
Chapter 6.
Gastrulation in Birds
We have already traced the establishment of the blastula (blastodisk) in the chick
embryo as a two-layered structure consisting of an upper layer (the epiblast) and a lower layer
(the hypoblast), with a thin blastocoel in between (Fig. 5-10). The embryo proper occupies the
transparent area pellucida and is surrounded by the area opaca, where the cells of the
blastoderm lie unseparated from the yolk (Fig. 6-15).
Figure 6-15: Chick embryos showing four stages in the formation of the primitive
streak. (A) 3 to 4 hours’ incubation; (B) 5 to 6 hours’ incubation; (C) 7 to 8 hours’
incubation; (D) 10 to 12 hours incubation. (Based in part on the photomicrographs of
Spratt, 1946, J. Exp. Zool., vol. 103.)
Gastrulation and formation of the definitive embryonic germ layers begin with the
appearance of a condensation of cells in the posterior part of the epiblast. This condensation,
seen in an embryo that has been incubated for 3 to 4 hours (Fig. 6-15a), gradually assumes an
anterior-posterior elongation (Fig. 6-15b). By the seventh or eighth hour of incubation, the
elongation is still more definite (Fig. 6-15c), and by the end of the first half day, the thickened
area has assumed a shape which has led to its being called the primitive streak (Fig. 6-15d).
The appearance of the primitive streak is the result of an inductive interaction of the
epiblast with the hypoblastic layer, and its orientation is a reflection of the intrinsic polarity of
pg. 2
the underlying hypoblastic layer. Recent experiments have shown that the organizational
center of the early chick embryo is located in the posterior margin of the hypoblast (Azar and
Eyal-Giladi, 1981).
The early primitive streak initially elongates in both an anterior and a posterior
direction. Throughout much of the posterior part of the blastoderm, cell movements
converge from the lateral areas toward the forming primitive streak (Fig. 6-16). As more cells
enter the streak region, the primitive streak elongates in a posterior direction. The anterior
extension of the primitive streak keeps pace with the expansion of the hypoblast beneath it.
pg. 3
After 16 hours of incubation the primitive streak becomes so prominent that embryos
are characterized as being in the primitive-streak stage (Fig. 6-17). A central furrow called the
primitive groove now runs down the center of the primitive streak. Along both sides it is
flanked by thickened margins, called the primitive ridges (Fig. 6-17). At the anterior end of the
primitive streak closely packed cells form a local thickening known as Hensen’s node (Fig. 6-17).
After the primitive streak has reached its full length at about the eighteenth hour of
incubation, the anterior end begins to regress, leaving in its wake a structure commonly
referred to as the head process. This is a gross morphological term referring to the area where
the notochord has been recently laid down (Figs. 6-16 and 6-17).
The part of the area pellucida adjacent to the primitive streak begins to thicken and is
said to constitute the embryonal area. Because of its shape, the embryonal area is frequently
spoken of as the embryonic shield. Accompanying the formation and elongation of the
primitive streak, the area pellucida undergoes a change in shape from an essentially circular
disk to a pear shaped configuration. The long axis of the future embryo is clearly established
by the primitive streak.
With the establishment of the primitive streak and Hensen’s node, the main period of
gastrulation begins. The embryonic germ layers are formed by the migration of cells in the
epiblast toward Hensen’s node and the primitive streak, and their ingression to form the
middle and lower germ layers (the mesoderm and endoderm). Ingression is the movement of
cells from the epiblast “down” into the blastocoel and the hypoblast. The anterior portion of the
primitive streak and the node serve as a passageway for cells even while the streak is
pg. 4
elongating anteriorly. Gastrulation is accomplished by the coordinated passage of individual
cells from the exterior into the interior of the embryo.
The first cells to pass through the area of the anterior part of the primitive streak are
future embryonic endodermal cells. After about 8 to 10 hours of incubation, more than 80
percent of these cells are found in the endoderm; the remainder migrate into the middle
mesodermal layer. As time goes on, a progressively greater percentage of the cells which pass
through the node are destined to be incorporated into the mesoderm and a correspondingly
smaller number lodge in the endoderm. The endodermal cells that are formed in this manner
enter the original hypoblastic layer and steadily displace the cells of the hypoblast outward
and anteriorly toward the edge of the area opaca (Fig. 6-18). Although the bulk of the
endoderm has passed through the nodal region during the early, formative stages of
development of the primitive streak, increasing numbers of future endodermal cells migrate
through the anterior part of the primitive streak as well. By about 22 hours of incubation,
when regression of the primitive streak has commenced, essentially all the future endodermal
cells have left the epiblast.
pg. 5
Little formative activity of the middle germ layer (embryonic mesoderm) occurs until
around the fifteenth hour of incubation, when the primitive groove becomes well established
within the primitive streak (Fig. 6-17). There are two principal areas of invagination and
mesoderm formation in the early chick embryo. The most extensive invagination of
mesodermal cells occurs along the length of the primitive streak, where the coherent layer of
mesodermal cells that is formed expands parallel to the underlying layer of the embryonic
endoderm (Fig. 6-21). The spread of the mesoderm is shown in Fig. 6-19. The other major
site of mesoderm formation is through Hensen’s node where a rod of mesodermal cells
directed anteriorly lies in the midline of the embryo in the track of the regressing primitive
streak. This mesodermal rod becomes the notochord (Figs. 6-19 and 6-21), which is essential
for neurulation. The mesodermal cells that leave the cranial part of the primitive streak
participate in the formation of embryonic mesoderm, whereas those that exit from the most
posterior part of the primitive streak become part of the extraembryonic mesoderm.
pg. 6
As the cells of the epiblast migrate toward and through the primitive streak and
ultimately take their place among the other cells of the mesodermal layer, they undergo
certain characteristic changes in form. The cells become bottle-shaped as a result of the
appearance of orderly arrays of intracellular microtubules, which are associated with changes
of shape in many varieties of cells. After they have passed through the primitive streak, the
cells of the mesoderm become connected to one another by small gap junctions (specialized
protein channels).
Shortly after the first notochordal cells are laid down, the primitive streak and Hensen’s
node undergo a regression toward the posterior end of the embryo. Accompanying this
regression is a corresponding elongation of the notochord. The expression of embryonic
genes can occur as early as the 16-32 cell stage in birds (similar to the mammalian pattern of
early embryonic gene expression).
pg. 7
Part 4:
Neurulation in Birds
and
Neurulation in Humans
pg. 1
Abridged/modified from: Carlson (1996) Foundations of Embryology, 6th ed, Chapter 7.
Neurulation in Birds
The morphogenetic movements during gastrulation not only result in the formation of
the three primary germ layers, but also cause groups of cells that were far apart in the
blastodisk to become located close to one another. The future developmental fate of the
embryo depends on inductive interactions among some of these newly associated groups of
cells. The primary inductive event is the action of the notochord on the overlying ectoderm,
resulting in the transformation of the unspecialized ectodermal cells into cells of the future
central nervous system. The initial response of the induced ectoderm is to form a plate of
thickened cells (the neural plate). Soon this plate becomes transformed into a longitudinal
groove and ultimately it folds up into a tube. While this is occurring, other ectodermal cells
from the junction between the neural and general cutaneous ectodermal tissues (destined to
become skin) form segmentally arranged aggregations that are known collectively as the
neural crest. Later, cells of the neural crest follow extensive and varied migration and
differentiation pathways throughout the body of the embryo.
Following the changes leading to the formation of the neural tube, the mesodermal
layer on either side of the notochord splits into longitudinal divisions. The blocks of
mesoderm on either side of the notochord soon begin to form symmetrical pairs of brick-like
masses called somites, which are both major landmarks in the early embryo and the source of a
number of important segmentally arranged mesodermal derivatives later in life (such as
vertebrae and ribs). The somite pairs first take shape near the cranial part of the embryo. In
successive stages additional pairs of somites are formed posterior to those already laid down.
From the earliest stages of formation of the nervous system, differentiation of axial structures
follows a pronounced anterior-posterior gradient. Because of these gradients, processes which
have already been completed in the anterior part of the embryo may be just beginning in the
posterior part.
Commonly, neurulation is considered to be the period of development starting with
the first traces of formation of the neural plate and ending with the closure of the neural tube.
Induction:
During late gastrulation, the notochord which forms from cells passing through
Hensen’s node in birds and mammals pushes anteriorly just beneath the ectoderm. While the
forward movement of the notochord cells is taking place, they induce the overlying
ectodermal cells, causing them to thicken and form the neural plate. This reaction, which both
initiates the formation of the central nervous system and causes the central longitudinal axis of
the body to be established, is commonly called neural induction. The inductor is the
notochordal tissue and the responding tissue is the ectoderm.
As in other inductive systems, it is essential that the inductor and the responding tissue
be at the right place at the right time. Without the presence of the underlying notochord, the
cells of the dorsal ectoderm do not form neural tissue but rather continue to differentiate as
pg. 2
general cutaneous ectoderm (skin). This has been demonstrated experimentally by
transplanting to the ventral side of the embryo small pieces of prospective neural ectoderm
before it has been acted upon by the notochord. The explants do not form neural tissue (Fig.
7-2). However, if the same operation is performed in the late gastrula, the grafted ectoderm
forms a neural plate, as it would have done if it had remained in its original location.
A
B
In order for neural induction to occur, the ectoderm overlying the notochord must be
able (competent) to respond to the inductive stimulus. During much of the period of
gastrulation, both the dorsal and ventral ectoderm have the competence to form neural tissues
when subjected to the influence of inductors. However, by late in the neurula stage most nonneural ectoderm has lost its neural competence.
For many decades after the discovery of the organizer (i.e. Hensen’s Node), the nature
of the inductive stimulus remained obscure. Only recently, a protein, called noggin, which has
no similarity to any other known inducing factor, has been demonstrated to fulfill the
requirements of the neural inductor in Xenopus (Lamb et al., 1993). The noggin gene is
expressed in notochord and the noggin protein is capable of converting ectoderm to neural
ectoderm.
pg. 3
Formation of the neural tube:
The neural plate does not remain flat very long. Soon after it has taken shape, its lateral
borders become elevated, forming the neural folds, which flank the neural groove (Fig. 7-10).
The two lateral edges of the neural folds eventually come together in the dorsal midline to
form a complete neural tube (Fig. 7-10). Closure of the neural tube first occurs in the upper
spinal cord levels and from there proceeds both anteriorly and posteriorly.
The mechanism of neural-tube formation has been the subject of much speculation over
the years, and even now not all aspects of the process are well understood. Modern
investigations have confirmed earlier speculations that at least part of the process of neural
folding can be attributed to intrinsic changes in shape of the neuroepithelial cells. Elongation
of a neuroepithelial cell requires the presence of a series of intact microtubules running from
the base to the apex of the cell (Fig. 7-11). The microtubules act like an internal skeleton,
supporting its greatly increased height. Meanwhile, just beneath the apical surfaces of these
cells are organized bundles of thin microfilaments, which can contract, resulting in the
constriction of the apical end of the cell (Fig. 7-11).
pg. 4
Changes in cell shape alone are insufficient to account for the formation of the neural
tube. Recent investigations in chick embryos have shown that neural tube formation is the
result of a number of factors acting in concert (Schoenwolf, 1990). Four main stages have been
identified in the formation of the avian neural tube. The first stage is the transformation of the
embryonic ectoderm into a thickened neural plate through neural induction. The second stage
consists of shaping the overall contours of the neural plate through intrinsic cell
rearrangements and region-specific changes in the shape of the neuroepithelial cells.
A third stage in avian and most probably mammalian neural tube formation is lateral
folding of the neural plate around a median hinge point. Bending at the hinge point can be
accounted for to a great extent by changes in cell shape, with apical narrowing forming a
wedge (Fig. 7-11). This area then acts as an anchoring point, as the two flatter sides of the
neural plate become elevated at a sharp angle from the horizontal. Elevation of the lateral
portions of the neural plate is accomplished largely by factors extrinsic to the neuroepithelium,
especially forces generated by the surface epithelium lateral to the neural plate.
The fourth stage of neural tube formation consists of apposition of the lateralmost
apical surfaces of the neural plate, with their fusion into a tube. This is followed by separation
of the neural tube from the general cutaneous ectoderm, which now covers the entire dorsal
surface of the embryo. While the neural tube is closing, a population of ectodermal cells
separates itself, forming the neural crest.
pg. 5
Adapted from: Gilbert (2000), Developmental Biology, 6th Ed.
pg. 6
Mammalian Neurulation:
Unlike neurulation in chicks (in which neural tube closure is initiated at the level of the
future midbrain, and “zips up” in both directions), neural tube closure in mammals is initiated
at several places along the anterior-posterior axis. Different neural tube defects are caused
when various parts of the neural tube fail to close (Fig. 12.6). Failure to close the human
posterior neural tube regions at day 27 (or the subsequent rupture of the posterior neuropore
shortly thereafter) results in a condition called spina bifida, the severity of which depends
pg. 7
on how much of the spinal cord remains exposed. Failure to close the anterior neural tube
regions results in a lethal condition, anencephaly. Here, the forebrain remains in contact with
the amniotic fluid and subsequently degenerates. Fetal forebrain development ceases, and the
vault of the skull fails to form. The failure of the entire neural tube to close over the entire
body axis is called craniorachischisis. Collectively, neural tube defects are not rare in humans,
as they are seen in about 1 in every 500 live births. Neural tube closure defects can often be
detected during pregnancy by various physical and chemical tests.
Human neural tube closure requires a complex interplay between genetic and
environmental factors. Certain genes, such as Pax3, sonic hedgehog, and openbrain, are essential
for the formation of the mammalian neural tube, but dietary factors, such as cholesterol and
folic acid, also appear to be critical. It has been estimated that 50% of human neural tube
defects could be prevented by a pregnant woman’s taking supplemental folic acid (vitamin
B12), and the U.S. Public Health Service recommends that all women of childbearing age take
0.4 mg of folate daily to reduce the risk of neural tube defects during pregnancy (Centers for
Disease Control, 1992).
pg. 8