GASTRULATION:

CLEAVAGE AND GASTRULATION IN MAMMALS

Birds and mammals are both descendants of reptilian species. It is not surprising, therefore, that mammalian
development parallels that of reptiles and birds. The gastrulation movements of reptilian and avian embryos, which
evolved as an adaptation to yolky eggs, are retained in the mammalian embryo even in the absence of large amounts of
yolk. The mammalian inner cell mass can be envisioned as sitting atop an imaginary ball of yolk, following
instructions that seem more appropriate to its reptilian ancestors.
• Mammalian egg is the smallest among the animal kingdom; the human zygote is 100 micrometer in diameter.
• Fertilization occurs in the oviduct, meiosis of egg complete after entry of the sperm.
• Cleavage after one day, cleavage is slow and takes 12 to 24 hours.
• As the zygote reaches the uterus, cleavage occurs along the journey.
• The first cleavage is meridional, and the second cleavage is rotational. The 2 blastomeres divide in different
planes (one is equatorial and one is meridional.)

Early Development in Mammals

Mammalian eggs are among the smallest in the animal kingdom, making them difficult to manipulate experimentally.
The human zygote, for instance, is only 100 µm in diameter—barely visible to the eye and less than one-thousandth
the volume of a Xenopus laevis egg. Also, mammalian zygotes are not produced in numbers comparable to sea urchin
or frog zygotes; a female mammal usually ovulates fewer than 10 eggs at a given time, so it is difficult to obtain
enough material for biochemical studies. As a final hurdle, the development of placental mammalian embryos is
accomplished inside another organism rather than in the external environment (although early embryos prior to
implantation can be cultured and observed in vitro). Most research on mammalian development has focused on the
mouse, since mice are relatively easy to breed, have multiple progeny in their litters, and are easily housed in
laboratories.

Mammalian cleavage
Prior to fertilization, the mammalian oocyte, wrapped in cumulus cells, is released from the ovary and swept by the
fimbriae into the oviduct. Fertilization occurs in the ampulla of the oviduct, a region close to the ovary. Meiosis is
completed after sperm entry, and the first cleavage begins about a day later. The positioning of the first cleavage plane
may depend on the point of sperm entry, and in mice, a sperm-borne microRNA (miRNA-34c) is required to initiate
this first cell division. This miRNA appears to bind and inhibit Bcl-2, a protein that prevents the cell from entering the
S phase of the cell cycle (Liu et al. 2012). The two nuclei produced by this cleavage are the first nuclei to contain the
entire genome, since the haploid pronuclei enter cell division upon meeting.

FIGURE - Development of a human embryo from fertilization to implantation. Compaction of the human embryo
occurs on day 4, at the 10-cell stage. The embryo “hatches” from the zona pellucida upon reaching the uterus. During
its migration to the uterus, the zona prevents the embryo from prematurely adhering to the oviduct rather than
traveling to the uterus.

THE UNIQUE NATURE OF MAMMALIAN CLEAVAGE Cleavages in mammalian eggs are among the slowest
in the animal kingdom, taking place some 12–24 hours apart. The cilia in the oviduct push the embryo toward the
uterus, and the first cleavages occur along this journey. In addition to the slowness of cell division, several other
features distinguish mammalian cleavage, including the unique orientation of mammalian blastomeres relative to one
another. In many but not all mammalian embryos, the first cleavage is a normal meridional division; however, in the
second cleavage, one of the two blastomeres divides meridionally and the other divides equatorially (FIGURE 12.11).
This is called rotational cleavage.
 FIGURE - Comparison of early cleavage in (A) echinoderms
and amphibians (radial cleavage) and (B) mammals (rotational
cleavage). Nematodes also have a rotational form of cleavage,
but they do not form the blastocyst structure characteristic of
mammals.

Another major difference between mammalian cleavage and that of anamniotes is the marked asynchrony of early
cell division. Mammalian blastomeres do not all divide at the same time. Thus, mammalian embryos do not increase
exponentially from 2 to 4 to 8 cells, but frequently contain odd numbers of cells. Furthermore, the mammalian
genome, unlike the genomes of rapidly developing animals, is activated during early cleavage, and zygotically
transcribed proteins are necessary for cleavage and development. Maternally encoded proteins can persist through
most of the cleavage stages and play important roles in early development. In the mouse and goat, the activation of
zygotic (i.e., nuclear) genes begin in the late zygote and continue through the 2-cell stage. In humans, the zygotic
genes are activated slightly later, around the 8-cell stage.

COMPACTION - One of the most crucial events of mammalian cleavage is compaction. Mouse blastomeres through
the 8-cell stage form a loose arrangement. At first, the blastomeres of mammalian embryos have a loose arrangement,
and touch only at the basal surfaces. After compaction, blastomeres adhere tightly, maximizing the area of contact
During compaction, each blastomere undergoes polarization. Tight junctions develop on the outer surface, allowing
proteins to specialize.
Following the third cleavage, however, the blastomeres undergo a spectacular change in their behavior. Cell adhesion
proteins such as Ecadherin become expressed, and the blastomeres gradually huddle together and form a compact ball
of cells. This tightly packed arrangement is stabilized by tight junctions that form between the outside cells of the ball,
sealing off the inside of the sphere. The cells within the sphere form gap junctions, thereby enabling small molecules
and ions to pass between them (much like in the fish early blastula).
After compaction at the 8-16 cell stage, there are 2 types of blastomeres. Outside blastomeres are tightly joined and
number about 9-14. They surround 2-7 inside blastomeres that are loosely joined.

The cells of the compacted 8-cell embryo divide to produce a 16-cell morula. The morula consists of a small group of
internal cells surrounded by a larger group of external cells. Most of the descendants of the external cells become
trophoblast (trophectoderm) cells, whereas the internal cells give rise to the inner cell mass (ICM).
The inner cell mass, which will give rise to the embryo, becomes positioned on one side of the ring of trophoblast
cells; the resulting blastocyst is another hallmark of mammalian cleavage.

The trophoblast cells produce no embryonic structures, but rather form the tissues of the chorion, the extraembryonic
membrane and portion of the placenta that enables the fetus to get oxygen and nourishment from the mother.
The chorion also secretes hormones that cause the mother’s uterus to retain the fetus, and it produces regulators of the
immune response so that the mother will not reject the embryo.
It is important to remember that a crucial outcome of these first divisions is the generation of cells that attach the
embryo to the uterus. Thus, formation of the trophectoderm is the first differentiation event in mammalian
development. These cells adhere to uterine lining, then digest a path and allow the embryo to lodge itself in the uterine
wall. Trophoblast gets differentiated into cytotrophoblast and syncytiotrophoblast
The earliest blastomeres (such as each blastomere of a 2-cell embryo) can form both trophoblast cells and the embryo
precursor cells of the ICM. These very early cells are said to be totipotent while the cells of the inner cell mass are
said to be pluripotent .
CAVITATION - In mice, the embryo proper is derived from the inner cell mass of the 16-cell stage, supplemented by
cells dividing from the outer cells of the morula during the transition to the 32-cell stage. The cells of the ICM give
rise to the embryo and its associated yolk sac, allantois, and amnion. By the 64-cell stage, the ICM and the trophoblast
cells have become separate cell layers, neither of which contributes cells to the other group.
The ICM actively supports the trophoblast, secreting proteins like fgf4 that stimulate the trophoblast cells to divide.

Initially, the morula does not have an internal cavity. However, during a process called cavitation, the trophoblast
cells secrete fluid into the morula to create a blastocoel. The membranes of trophoblast cells contain sodium pumps
that pump Na+ into the central cavity. The subsequent accumulation of Na+ draws in water osmotically, creating and
enlarging the blastocoel.

During gastrulation, ICM forms two layers. The upper layer of ICM is epiblast and the lower layer is hypoblast.
The hypoblast migrates and lines the blastoceol cavity, and later give rise to extra embryonic endoderm.

The epiblast cell layer is split by small clefts that eventually coalesce to separate the embryonic epiblast from the other
epiblast cells that form the amnion. Once the amnion is completed, the amniotic cavity fills with amniotic fluid, a
secretion that serves as a shock absorber and prevents the developing embryo from drying out. The embryonic epiblast
is thought to contain all the cells that will generate the actual embryo and is similar in many ways to the avian epiblast.
Gastrulation begins at the posterior end of the embryo, and this is where the primitive streak arises
Mammalian mesoderm and endoderm cells originate in the epiblast, undergo an epithelial-mesenchymal transition,
lose E-cadherin, and migrate through the primitive streak as individual mesenchymal cells. Cells which migrate forms
the mesoderm and endoderm, due to fgf8.

THE ROLE OF FGF IN EARLY MOUSE GASTRULATION

Cell migration and specification are coordinated by fibroblast growth factors. The cells of the primitive
streak appear to be capable of both synthesizing and responding to FGFs. In embryos that are homozygous
for the loss of the Fgf8 gene or its receptor, cells fail to emigrate from the primitive streak, so a very thick
streak develops and neither mesoderm nor endoderm are formed. This suggests that the main function of
FGF8 (and its receptors) is to drive the mesendoderm out of the streak, perhaps by chemorepulsion, as has
been suggested in chick. Fgf8 may also control cell specification by regulating snail, Brachyury, and Tbx6,
three genes that are essential (as they are in the chick embryo) for mesodermal migration, specification, and
patterning. The ectodermal precursors are located anterior and lateral to the fully extended primitive streak,
as in the chick epiblast; also, as in the chick, a single cell can give rise to descendants in more than one germ
layer. Thus, at the epiblast stage, these lineages have not yet become fully separate from one another.
Indeed, in mice, some of the visceral endoderm, which had been extraembryonic, is able to intercalate with
the definitive endoderm and become part of the gut.
Mechanism of cleavage-
Cleavage is a result of two process -
1. Cyclin process- Karyokinesis, mitotic division of nucleus. Spindle fibre composed of microtubules is the
mechanical agent.
2. 2nd process is cytokinesis, mechanical agent is contractile ring or microfilament made of actin.
The processes of karyokinesis (mitosis) and cytokinesis work together to result in cleavage. The mitotic apparatus is
made up of a central spindle and polar asters made up of polymers of tubulin protein called microtubules. The asters
are nucleated by centrosomes and the centrosomes are organized by centrioles brought into the egg by the sperm as
basal bodies. Cytokinesis is mediated by the contractile ring made up of polymers of actin protein called
microfilaments. Karyokinesis and cytokinesis are independent but spatially and temporally coordinated processes.
While mitosis can occur in the absence of cytokinesis, cytokinesis requires the mitotic apparatus. Contractile ring
creates cleavage furrow which eventually bisects the plane of mitosis, thereby creating two genetically equivalent
blastomeres.
After fertilization multicellular organisms are produced by cleavage of mitotic division, an enormous volume of
cytoplasm is divided into many small nucleated cells called blastomeres. Rate of cleavage depends on proteins and
mRNA stored in the egg cell. During cleavage cytoplasm volume is not increased, but cytoplasm present in zygote is
distributed to blastomeres. Zygote divides into half, quarters, eighths and so forth. Division of egg cytoplasm without
increasing is possible by omitting growth period between cell division, that is G1 and G2 phases. Same way the
nucleus also divides at a high rate never seen in tumour cells. Ex: frog egg can divide 37,000 cells in 43 hours, in
Drosophila 50,000 cells in two hours.