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Name:______________________________________________________ Date Performed/Submitted:_________

Section/ Group Name:_________________________________________ Score:_________________________

Exercise 3
Gastrulation
Introduction:
During gastrulation, highly coordinated cell movements transform the blastula from a ball of
blastomeres to a highly organized embryo called a gastrula with three distinct layers from which all organs and
tissues originate. The three layers of the gastrula are collectively called embryonic germ layers, and include the
ectoderm, mesoderm, and endoderm. While presumptive (future) ectoderm cells remain on the exterior of the
gastrula, blastomeres that will become mesoderm and endoderm migrate internally. The new locations of these
cells allow tissues and organs to form in their proper positions in a process called organogenesis. The ectoderm
gives rise to the epidermis and components of the nervous system, the endoderm generates the epithelium of
the gut and gut derivatives (e.g., liver, pancreas), and the mesoderm forms the circulatory system, muscles,
kidneys, gonads, dermis, and skeletal components. At the end of gastrulation, the gastrula is characterized by
an inner layer of endoderm, an outer layer of ectoderm and the middle layer of mesoderm. Understanding how
cells differentiate into the three germ layers and exhibit specialized movements is one of the major areas or
research in developmental biology today. This exercise explores the process of gastrulation using several model
organisms to demonstrate different gastrulation strategies.

Basic Cell Movements

Gastrulation movements involve the entire embryo. Cell movements of any area in the gastrula are
synchronized with movements of other areas of the embryo. The pattern of gastrulation can vary amongst
different species but the embryo exhibit similar types of basic cell movements.
Invagination: infolding or buckling inward of a sheet of cell;
analogous to a finger poking a balloon (e.g. sea urchin ectoderm).
Involution: inward migration of an expanding outer sheet of cells
so that it spreads over the basal (internal) surface of the remaining
external cells (e.g. amphibian mesoderm).
Ingression: migration of individual cells into the embryo's
interior (e.g. urchin mesoderm).
Delamination: splitting of a single sheet of cells into two separate
but parallel sheets (e.g. chick and mammalian hypoblast
formation).
Epiboly: expansion of an outer sheet of cells by mitosis to cover the entire embryo's surface; movement
is analogous to syrup being poured over an orange (e.g. ectoderm in sea urchins, tunicates, amphibians,
fish, and birds).
Convergence: migration of cells toward a common point such as the midline of the embryo (e.g. cell
migration over the frog dorsal blastopore lip, and chick primitive streak formation).
Extension: rearrangement and elongation of a cluster of cells to form a thin, stretched cellular group.
Analogous to a group of people that form a large crowd while waiting for a bus, but move in single file
when boarding it (e.g. sea urchin endoderm during archenteron formation, and cell migration over the
frog dorsal blastopore lip.

Sea Urchin Gastrulation

Sea urchins exhibit a simple type of gastrulation that occurs soon after the blastula hatches from the
fertilization envelope. At the onset of gastrulation, primary mesenchymal cells (derived from micromeres) ingress
into the blastocoel, and later differentiate into mesoderm. The vegetal side of the blastula flattens into a vegetal plate
that invaginates forming the archenteron (embryonic gut, also called gastrocoel). The opening of the archenteron is
the blastopore, which in sea urchins is the site of the future anus. The archenteron elongates by convergent extension,
reaches the inner surface of the blastocoels wall, and fuses with the ectoderm to form the stomodeum (mouth). The
anus marks the future dorsal side (aboral side) and the stomodeum forms on the ventral side (oral side).
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The gastrula next progresses

through the prism and pluteus larval
stages. The prism stage does not have
larval arms, whereas the pluteus has
two pairs: a prominent long ventral
pair (anal or dorsal arms), and a
short dorsal pair: near the mouth
(oral or ventral arms). Spicules
made of calcite (CaCO3, MgCO3)
provide skeletal supports for the
larval arms. At the prism stage, the
spicules begin to lengthen giving the
embryo a "prism" shape. Further
spicule elongation enables larval
arms to grow out in the pluteus. The
archenteron differentiates into three
regions of the gut (esophagus,
stomach, intestine) which appears
shaped like J in the pluteus. The
pluteus is covered with cilia which
allow it to swim freely with its arms
forward. The pluteus will later
metamorphose into a small sea urchin, which grows spines, becomes sedimentary and resembles the adult form.

Frog Gastrulation
The pigmented animal hemisphere of amphibian embryos (e.g Rana pipiens, Xenopus laevis) makes cell
movements during gastrulation visible as they drastically change the embryos appearance. Gastrulation begins on
the dorsal side in the gray crescent at the marginal zone, an area surrounding the equator of the embryo where
micromeres and macromeres meet. A group of cells (bottle cells) in the dorsal marginal zone constrict at one end,
becoming bottle-shaped and invaginate to form the
leading edge of the archenteron. This causes a
pigment difference on the embryo's surface which
becomes visible as the dorsal lip of the blastopore,
over which marginal zone cells (future mesoderm)
involute. In some frogs, the archenteron roof is
initially lined with involuted mesoderm
(chordamesoderm) that later becomes covered with
endoderm. The archenteron floor is lined with
vegetal cells that become endoderm. The blastocoel
is displaced as the archenteron enlarges. Eventually
the blastopore lip widens into a crescent forming
lateral and ventral lips as additional marginal zone
cells involute. The dorsal lip of the blastopore has
been called the frog organizer (Spemann's
organizer), since it induces cell fates and "organizes" cells into specific tissues. It is analogous to the fish
embryonic shield. The blastopore is the site of the future anus in amphibians. Exterior cells of the animal
hemisphere proliferate and move by epiboly to cover the entire embryo's surface with pigmented micromeres.
Near the end of gastrulation, the dark brown embryo has a small, light yellow yolk plug (patch of endoderm cells
that has not yet been covered by pigmented cells, and is bounded by the lips of the blastopore). Internalization of
the yolk plug by epibolic movements of the ectoderm signals the end of gastrulation.
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Fig. 3.4 Sagittal section of frog

gastrulae: late blastula (A-B); early
gastrula with dorsal lip (C); mid gastrula
with ventral lip and yolk plug (D); late
gastrula with yolk plug (E-F).

I =fertilization envelope
2=animal pole
3 = blastocoel
4=vegetal pole
5=dorsal lip of blastopore
6=archenteron
7=blastopore
8=yolk plug
9=ventrol lip of blastopore
10=outer layer of ectoderm
11 =inner layer of ectoderm
12=chordamesoderm (roof of
archenteron)
Double-headed arrow= marginal
zone
Avian Gastrulation
Avian gastrulation begins in the embryo's thickened caudal margin (marginal zone or belt) where the
area pellucida and area opaca meet. The direction of cell movements during gastrulation is shown in Fig. 3.5.
Cells migrate into the embryo through the primitive streak, a midline thickening of epiblast located in the caudal
two-thirds of the embryo. Primitive folds (primitive ridges) flank a midline depression down the primitive streak
called the primitive groove. Future endoderm cells are initially located in the epiblast, but later ingress through
the primitive streak to populate the hvpoblast. Prospective mesoderm cells migrate through the primitive streak
and populate the area between the epiblast and hypoblast. Cells that ingress through the primitive pit (most rostral
region of primitive groove) form: 1) a thickened area rostrally called Hensen's node (primitive knot), and 2) the
head process which becomes the anterior portion of the notochord. Hensen's node is the avian "organizer."
The primitive streak is first visible from 6-10 hours of incubation after egg-laying. It elongates and reaches its full
length by 18 hours of incubation. The primitive streak then regresses in the cranial region of the embryo as future
mesoderm and endoderm cells migrate through it. The primitive streak is visible only in the caudal region of the
33-hour embryo stage (stage 10), and has regressed completely by 48 hours.
Fig. 3.5 shows the cut away view of a chick gastrula showing migration of presumptive mesoderm (red)
and endoderm (yellow) cells through the primitive streak. Arrows show the direction of migration from the
epiblast (blue) to the blastocoel (gray) and hypoblast (purple).Cells remaining in the epiblast after gastrulation are
ectoderm. Migrating endoderm cells eventually replace the hypoblast. Dorsal lip is up.

Fig. 3.5 Cut away view of a chick gastrula .

1= epiblast, 2= hypoblast, 3= subgerminal cavity, 4= developing neural tube,

5= head process, 6= ingressed endoderm, 7= ingresses mesoderm, 8= Hensens
node, 9= primitive groove, 10= primitive groove, 11= area opaca, 12= yolk

Fig. 3.7 Transverse sections through a 16-hour chick embryo: A) cranial

blastoderm; B) head fold (rostral notochord); C) Hensen's node; D) rostral
primitive streak; E) caudal primitive streak; F), caudal blastoderm. Ln the
cranial and caudal blastoderm (A, F), the bilaminar appearance of the
blastoderm is demonstrated, and the subgerminal cavity is visible. The epiblast
is thicker in the head fold, Hensen's node, and primitive streak. Ingressing cells
migrate over the primitive folds which are raised above the blastoderm surface.
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Fig. 3.6 Whole mount of 16-hour chick embryo.

Materials:
Microscope
Prepared slides
Sea Urchin: early gastrula, late gastrula, pluteus
Starfish: early gastrula
Frog: early gastrula, late gastrula

Procedure:
1. View slides under scanner, LPO and HPO.
2. Sketch/draw observed slides (under HPO). Label the parts.

Question:
1. Discuss the importance of gastrulation in embryonic cell differentiation.
2. List and describe some developmental anomaly associated with gastrulation.

Name:_____________________________________________ Date Performed/Submitted:_________________

Section/ Group Name:________________________________ Score:__________________________________

Laboratory Worksheet
Exercise 3-Gastrulation
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Name:____________________________________________ Date Performed/Submitted:___________________

Section/ Group Name:_______________________________ Score:_____________________________________

Laboratory Worksheet
Exercise 3-Gastrulation
 6

Name:______________________________________________________ Date Performed/Submitted:_________

Section/ Group Name:_________________________________________ Score:___________________________

Exercise 4
Neurulation

Introduction:
One of the most intriguing features that distinguish chordates from other animals is the presence of the
hollow notochord. In vertebrates, the nerve cord expands into the central nervous system (brain and spinal
cord) with the brain cranial and spinal cord caudal. How does the nerve cord form? Neurulation is the process in
which the rudiment of the nerve cord, called the neural tube, forms and separates from the overlying surface
ectoderm. The neural tube is a hollow cylinder of neural ectoderm cells (neuroepithelium). An embryo
undergoing neurulation is called a neurula.
The ectoderm of the neurula differentiates into three major cell types: 1) skin ectoderm (epidermis' future
skin), 2) neural ectoderm (future central nervous system), and 3) neural crest (forms part of the peripheral
nervous system and several non-neuronal cell types). This exercise focuses on the neural ectoderm and the
process of neurulation by which it develops into the neural tube.
There are two types of neurulation: 1) primary neurulation in which the neural ectoderm folds to form a
hollow neural tube, and (2) secondary neurulation in which mesenchymal cells aggregate to form a solid neural
rod (medullary rod) that hollows our to form the neural tube. ln frogs, birds, and humans, most of the neural tube
forms by primary neurulation, and only the caudal region develops by secondary neurulation.

Chordate embryos that undergo primary neurulation use a general pattern (Fig. 4.1). First evidence of
neurulation appears on the embryo's dorsal side in ectoderm lying above the chordamesoderm. The
chordamesoderm is a group of condensed mesoderm cells that lie on the dorsal side of the embryo, between the
ectoderm and endoderm of the embryonic gut. The chordamesoderm differentiates into the notochord (a
longitudinal, rod-like structure that runs most of the length of the embryo between the digestive tube and the
nerve cord). In many species, the chordamesoderm/notochord produces inductive signals that allow the
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overlying ectoderm to differentiate into neural ectoderm. The neural ectoderm cells elongate into a columnar
shape, forming a flat, longitudinal plate of cells called the neural plate. The lateral ends of the neural plate rise up
into neural folds, giving the neural plate a U- or V-shaped appearance. Between the neural folds is a depression,
the neural groove. The raised neural folds move toward each other and fuse to form a hollow, cylindrical neural
tube that becomes covered in skin ectoderm. The neural tube's cavity is the neurocoel. The cranial and caudal tips
of the neural tube close last, and their open ends are called the cranial neuropore (rostral neuropore, anterior
neuropore) and caudal neuropore (posterior) respectively.

Fig. 4.1 Diagram showing primary neurulation in transverse sections

of chordate embryo. (A) Dorsal ectoderm overlying the
chordamesoderm (notochord) of a late gastrula embryo. (B) In
1 =epidermis contrast to presumptive cells of neural crest (dark green) and
2= neural crest epidermis (blue), neural ectoderm cells (light green) become
3=neural plate columnar and are visible as the neural plate (B), which bends
4= notochord forming the neural groove in (C). The neural folds are elevated,
5=neural fold lateral edges of the neural plate that fuse to their tips to form the
6=neural neural tube in (D) and (E). The epidermis covers the neural tube and
groove neural crest cells. Neural crest cells will later migrate laterally from
7=neural tube the neural tube area.
8= neurocoel

Frog Neurulation

Frog neural tube forms by primary neurulation. The notochord sends inductive signals to the overlying
dorsal ectoderm cells, which respond by becoming columnar in shape. The patch of columnar ectoderm cells will
become the future CNS and is the first visible as the pear-shaped neural plate. The wider anterior end will form
the brain, and the narrow posterior end will develop into spinal cord. At the mid-neurula stage, the neural plate
quickly elongates and simultaneously its lateral edges rise up above the ectoderm as neural folds that surround
the neural groove. By the late neurula stage, the neural folds fuse in the midline to form the hollow neural tube
and neurocoel. While the neural tube forms, it sinks into the embryo toward the notochord and becomes covered
with epidermis. The most posterior region of the spinal cord forms by secondary neurulation.

Fig. 4.1 Dorsal view of frog neurula at early neural fold/ neural groove stage and transverse section through the frog early neural
fold/neural groove stage (A-D).1= neural fold, 2= epidermal (skin) ectoderm, 3=neural groove, 4= neural plate, 5=neural crest, 6= head
mesenchyme, 7=endoderm, 8=prochordal plate, 9= archenteron, 10 paraxial mesoderm, 11= notochord, 12= endodermal yolk mass.

Fig. 4.2 Dorsal view of frog neurula at late neural fold/ neural groove stage and transverse section through the frog late neural fold/neural
groove stage (A-D). 1= neural fold, 2= epidermal (skin) ectoderm, 3=neural groove, 4= neural plate, 5=neural crest, 6= head mesenchyme,
7=endoderm, 8=foregut , 9= paraxial mesoderm, 10= notochord, 11= endodermal yolk mass, 12=lateral plate mesoderm, 13=liver
diverticum, 14= midgut, 14=hindgut.
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(1) (2
)

Fig. 4.5 Frog neurula at neural tube stage:

(1) Sagittal section of a frog neurula at the
neural tube stage showing the level of the
sections. Color scheme: ectoderm (blue),
neural ectoderm (green), mesoderm (red),
endoderm (yellow), neurocoel (dark green),
and embryonic gut (dark brown).
(2) Transverse sections (A-L). Dorsal faces
up. The neural tube encloses a cavity, the
neurocoel. Anteriorly, the optic vesicles
(rudiments of the eyes) lie on each side of
the prosencepalon. Dorsal to the paired
optic vesicles are the paired optic placodes,
(future inner ear).Only one otic placode is
visible in sections B and C, since the plane
of section did not cut through both optic
placodes simultaneously. In B, a thickening
of the ectoderm ventral to the
prosencephalon is the adenohypophysis
(anterior pituitary gland). Note the
notochord lie ventral to the neural tube in
sections C to J. Several structures of the
embryonic gut are visible as spaces in the
sections as they are traced from anterior to
posterior: the pharynx (C, D), foregut (E,
F), midgut with liver diverticulum (G, H),
and hindgut (l-K) which later connects to
the proctodeum (L). The ventro-posterior
portion of the neurula is filled with bright
pink, yolk-filled endoderm cells (G-K).
Legend: I =dorsal side of embryo,
2=epidermal (skin) ectoderm, 3 =
mesencephalon, 4=head mesenchyme, 5=
prosencephalon, 6=adenohypophysis,
7=opitic placode, 8=optic vesicle,
9=adhesive gland, 10=stomodeum,
11=rhombencephalon, l2=notochord,
13=pharynx I4= neural crest, I5=endoderm,
l6=foregut, l7=mesoderm, 18=spinal cord, l9=somite, 20=first pharyngeal pouch, 21 =pronephric swelling (nephrotome), 22=midgut,
23=liver diverticulum, 24=lateral plate mesoderm, 25= hindgut, 26=neurenteric canal, 27=proctodeum
Avian Neurulation

The epiblast is the site of neurulation in avian embryo. While the neural ectoderm undergoes neurulation,
the embryo also develops mesoderm and endoderm. Neurulation begins in the cranial region of the chick embryo
and gradually progresses caudally. Both gastrulation and neurulation occur simultaneouslv in different regions of
the chick embryo (e.g. neurula's cranial region undergoes neurulation (neural folds are visible), its caudal region,
has just begun gastrulation (primitive streak is present).
Because the blastoderm forms within the Hens oviduct, the extent of development can vary widely at
the time the egg is laid. Development stops (due to the cooler temperature outside the hens body) until the egg is
incubated. Significant variations in development occur by 24 hours of incubation. Thus referring to incubation
time is not a reliable indication of progress of early embryonic development. The staging system of Hamburger
and Hamilton uses the number of somites to distinguish between early developmental stages (from about 20-50
hours of incubation).
Development of the avian nervous system depends on the notochord. The prospective notochord cells are
first visible in the 18-hour chick embryo as the head process. The ectoderm overlying (dorsal to) the head process
develops into a broad neural plate of thickened neural ectoderm. The neural plate is distinguished from the
primitive streak by the presence of the notochord ventral to the neural ectoderm. As neurulation progresses, the
neural plate narrows and lengthens by convergent-extension.
The neural plate bends ventrally in the midline in 20-22-hour chick embryos to form the neural folds that
are separated by the neural groove. In the whole mount, neural folds appear as two parallel dark lines in the
cranial dorsal midline of the embryo.
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In transverse sections of the embryo's anterior, the neural folds are U- or V-shaped areas of thickened
neural ectoderm. As transverse sections of 21-hour chick embryos are traced caudally, the neural folds diverge as
they flatten into the neural plate. This also holds true for the 24-hour embryo. The neural folds overlie the somites,
but are obscured by them and are more visible in transverse sections. Figures 4.6-4.7 show, a range of
development in different chicken eggs incubated for 24 hours. The embryos have five to seven somite pairs. The
elevated neural folds will either soon meet in the dorsal midline or fuse to form the neural tube in the 5-somite
embryo, or the neural tube has already formed in the cranial region of the 6-somite embryo. At the rostral tip of
the neural tube, the neural folds are unfused leaving a temporary opening, the cranial neuropore that closes by
33 hours. By 48 hours, the neural folds have fused indicating that primary neurulation is complete. The notochord
is visible directly central to the neural ectoderm of the neural folds and tube.

Fig 4.6 Dorsal view of a 24-hour

chick embryo whole mount
showing section planes of Fig. 4.7

Fig. 4.7 Transverse sections of a 24-hour chick embryo. Dorsal is up. A is the most cranial of these sections and M is the
most caudal.

Materials:
Microscope
Prepared slides
Frog: neural tube, neural fold, neural groove

Procedure:
1. View slides under scanner, LPO and HPO.
2. Sketch/draw observed slides (under HPO). Label the parts.
 10

Questions:
1. List and describe some developmental anomaly associated with neurulation.
2. Describe the effect of primitive streak duplication.

Name:____________________________________________ Date Performed/Submitted:___________________

Section/ Group Name:_______________________________ Score:_____________________________________

Laboratory Worksheet
Exercise 4-Neurulation
 11

Name:________________________________________ Date Performed/Submitted:_______________________

Section/ Group Name:___________________________ Score:_________________________________________

Laboratory Worksheet
Exercise 4-Neurulation