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BIOLOGY 205/SECTION 7
DEVELOPMENT-LILJEGREN
Lecture 4
Gastrulation
2. Frog gastrulation: Similar to the sea urchin, but more complex.
e. Next, the MARGINAL ZONE CELLS (cells at the junction between the
animal and vegetal hemispheres) begin involution at 2 levels:
i. the outside cells involute to form the roof of the archenteron, which will
form the endoderm
ii. the deep or inside cells involute to form mesodermal derivatives. This
movement is dependent on fibronectin in the extracellular matrix,
which is secreted by the ectoderm of the blastocoel roof shortly before
gastrulation. This was determined by an experiment that involved
injecting a synthetic fibronectin peptide competitor into the blastocoel.
The mesodermal precursor cells bind the synthetic fibronectin competitor
so can’t recognize the normal fibronectin-lined traffic route along the
blastocoel roof. The archenteron fails to form and these mesodermal
precursors remain at the surface.
f. The dorsal mesoderm undergoes convergent extension starting halfway
through gastrulation. This pushes the blastopore lip ventrally.
g. The ANIMAL CAP (ectoderm) spreads over the embryo by epiboly, converging at
the blastopore.
h. Mesenchyme migration: mesodermal cells crawling along inner surface of
blastocoel presumably via interactions with extracellular matrix.
3. The three germ layers formed by gastrulation will produce all of the embryonic
structures except the germ line.
a. The ECTODERM is the most external layer and will produce skin & through later
invagination of neural tube-->central nervous system. In vertebrates, migrating
neural crest cells--> peripheral nervous system & many other structures,
including some bone, cartilage, and connective tissue in the head.
b. The MESODERM is the middle layer and will produce muscle, connective tissue,
bones, blood and blood vessels. In vertebrate also --> notochord (progenitor of
vertebrae), bones & cartilage, circulatory and urogenital systems (kidneys,
gonads).
c. The ENDODERM is the inner layer and will produce the gut (entire digestive tube
from mouth to anus) and internal organs such as liver, lungs, pancreas. Also -->
organs that arise as outpocketings of gut in vertebrates= lungs, liver, pancreas,
salivary glands.
4. Human gastrulation
a. Major modification in mammalian development results from the need for a
connection to mom.
b. Thus while cleavage leads to a hollow ball, only inner cell mass will become
the embryo. Trophoblast cells cells go on to make the placenta, chorion,
amnion.
c. Inner cell mass gastrulates AFTER formation of placental connection to mom.
d. Unlike in frog, individual epiblast cells move into primitive groove to form the
mesoderm and endoderm. cell movements = individual cells ingressing, then
migrating
e. Hensen’s node acts like the frog dorsal lip (organizer); primitive groove is like the
frog blastopore.
5. The dorsal/ventral axis in the frog
a. Mother sets up initial axis of polarity: animal & vegetal poles
b. sperm entry is the critical cue in setting up the dorsal-ventral axis and results
in formation of the blastopore 180° opposite sperm entry point. How does
this work?
c. Sperm enters randomly at one side of the animal hemisphere
1.) Entry of sperm nucleus leads to cytoskeletal rearrangements which lead to
30° rotation of outer cortex relative to inner cytoplasm.
2.) This rotation leads to juxtaposition of animal pole cytoplasm with vegetal pole
cortex, and forms an area called the gray crescent. The gray crescent region in
the fertilized egg marks the future site of the dorsal blastopore lip in the later
embryo starting to undergo gastrulation.
6. Experiments in Gastrulation
a.
Experiment #1- determine basis of gray crescent formation
i. Rotate egg so that sperm entry point is now on top.
ii. Prevents normal 30° cortical rotation.
iii. As a result:
1. Inner cytoplasm shifts due to effects of gravity
2. New region of juxtaposition of animal pole cytoplasm -- vegetal
pole cortex next to sperm entry point
3. Blastopore forms there instead.
Conclusion: juxtaposition of animal pole cytoplasm to vegetal pole cortex
is critical factor!
b.
Experiment #2—Dorsal blastopore lip is the organizer.
most famous transplantation experiment that resulted in a Nobel prize. Hans
Spemann and Hilde Mangold published this in 1924 from Mangold’s doctoral
thesis. Transplant dorsal blastopore lip of newt embryo (forms at gray crescent
region) to another region of recipient embryo. Causes the formation of two
blastopores and a double embryo. They named this region the Organizer, since
it can direct formation of gastrulation and an entire new axis.
c.
Experiment #3—Induction of dorsal blastopore lip in
Xenopus.
1.
Start with 2 embryos, one normal, one irradiated
(UV irradiation prevents gastrulation). Transplant dorsal vegetal
blastomeres (which underlie the prospective dorsal lip region) of normal
embryo into irradiated embryo. In an irradiated embryo that didn’t
undergo transplantation, would just get ventral ‘belly piece’ since
gastrulation failed to occur. If do transplant, get normal embryo.
2. If transplant dorsal vegetal blastomeres of normal embryo into ventral
side of another normal embryo, get a 2nd blastopore and axis in addition
to original.
Conclusion: Dorsal vegetal cells underlying the prospective dorsal lip induce
them to initiate gastrulation.
Neurulation
1.
Neurulation is the formation of the nervous system.
a.
Many of the same cellular processes and cell shape changes that occur during
gastrulation are involved in neurulation, such as invagination and convergent
extension.
b.
In animals, nervous system & epidermis both derived from ectoderm--i.e.
CNS is derived by invagination of subset of ectoderm.
c.
In vertebrates, this entire process of setting aside precursors of nervous
system= neurulation
2.
The PROCESS of neurulation.
a. Dorsal ectodermal cells divided into 3 sets.
i. Dorsal-most are cells (purple in ppt) called the neural plate which will go
on to form the neural tube
ii. Adjacent to the neural plate are the cells (green) which will form the
neural crest.
iii. Even more ventral cells (blue) go on to form the epidermis
b. The neural plate forms - epidermal cells change their cell shape and become
more columnar.
c. central neural plate cells (purple) constrict apically, leading to invagination
d. neural plate begins to buckle, creating a neural fold with a neural groove in the
middle. This process along with neural plate formation result in part from shape
changes in cells. Convergent extension is important.
e. The neural folds elevate and converge at the midline to begin neural tube
closure.
f. Eventually the neural tube pinches off to become a separate structure, the
epidermis above it fuses, and the neural crest cells at the fusion point (green)
leave epithelium and migrate to become peripheral nervous system and skin
pigment cells.
g. Neural tube closure usually starts in the middle and moves both anterior and
posterior.
h. IF the neural tube does not close properly on the posterior side, the condition is
spina bifida. IF neural tube does not close properly on the anterior side, a fatal
condition called anencephaly can occur that severely affects brain development
(usually forebrain is absent). Neural tube defects are not rare (1 in every 500
live births). Folic acid is crucial for neural tube closure, and it is recommended
that all women of childbearing age take 0.4 mg of folate daily because the
development and closure of the neural tube are normally completed within 28
days after conception, before many women are aware that they are pregnant.
i.
Changes in cell adhesion properties help the neural tube separate from the
ectoderm. The outer ectoderm expresses E-cadherin, while the invaginating
neural plate ceases E-cadherin expression and instead expresses N-cadherin.
When the neural tube has formed, the neural crest cells between it and the
ectoderm express neither of these cadherins.
Induction
1) In the first lecture, we talked about how cells can get information from neighbors which
can influence their cell fate. Today we’ll be talking more in detail about the kind of
signaling that can communicate information between cells.
a) INDUCTION is the process whereby one group of cells provides information to a second group
of cells, and this information specifies or influences the FATE of the second group of cells.
b) To have an induction we need two components:
i)
an INDUCER, the cells that produce a signal (ie. small protein or ligand) that will change
the behavior of other cells.
ii) a RESPONDER, the cells that respond to the signal from the inducer.
c) Not all cells can respond to signals from the inducer: the ability to respond to a certain
inductive signal=COMPETENCE. The responder cells must be competent to receive the
inducing signal (ie: express a receptor)
d) By successive inductions it is possible to generate many different cell types from a few
interactions. Ie. induced cells (cells that were responders) that have made a cell fate decision
can turn around and induce other tissues, or even the cells that were the original inducers.
e) The cell movements involved in gastrulation provide cells with many new neighbors and
thus many opportunities for inductions.
2) Review of different types of signaling and signaling molecules.
a) Signaling molecules can be proteins or small molecules
b) Signals can act
i) Very short range- cells in direct contact.
ii) Locally among neighbors, ie. by secreted molecules that travel a short distance.
iii) Globally throughout the body. Ie. hormones produced by pituitary act in the ovary on
developing follicles.
iv) In a graded fashion. Ie. strong signal close by, weaker signal farther away. If high levels
of signal, get one cell fate. If low levels, get a different cell fate.
c) Induction allows an initial difference to be amplified into many cell types
3) Local Signaling: MESODERM INDUCTION in the frog embryo.
Initial asymmetries of Xenopus eggs (e.g. animal & vegetal poles) lead to some cell fate
differences, but not all fates can be created this way! These cell fates arise from cell:cell
interactions. e.g. Mesoderm arises at ectoderm/endoderm junction. Top half (animal pole) of
frog blastula is fated to become ectoderm (top) and mesoderm (equatorial part), while bottom
half (vegetal pole) is fated to become endoderm. We’re going to go over a series of three
experiments that demonstrated how induction of the mesoderm occurs:
i) Experiments: Neither vegetal nor animal pole alone can make mesoderm.
(1) If you separate animal (animal caps) and vegetal parts
(2) Culture them separately
(3) Result: Vegetal pole cells become endoderm and Animal pole cells become ectoderm,
but no mesoderm forms. [If you isolate animal cells that were located in the marginal
zone and did come in contact with vegetal cells before you removed them, they can
make mesoderm when cultured separately]
(4) If you culture animal cap and vegetal cells together, vegetal cells still form endoderm,
but the animal caps can be induced to form mesoderm instead of ectoderm. Thus,
vegetal pole cells can induce animal pole cells to make mesoderm
(5) THEREFORE A SIGNAL IN THE VEGETAL PART IS NEEDED TO INDUCE MESODERM IN
THE ANIMAL CAPS.
ii) These experiments led to the 3-step MODEL OF MESODERM INDUCTION.
(1) Dorsal-most vegetal cells induce dorsal animal pole cells above them to become the
“organizer" (and also dorsal mesoderm). [These dorsal vegetal cells able to induce
formation of the dorsal mesoderm were named the Nieuwkoop center after a Dutch
scientist Pieter Nieuwkoop who was involved in these experiments in the 1960s and
70s.]
(2) More ventral vegetal cells induce other animal pole cells to become ventral +
intermediate mesoderm
(3) The Organizer (dorsal margin cells) induces the neighboring margin zone cells to
become intermediate mesoderm.
iii) What are the molecular pathways responsible for mesoderm induction?