Gastrulation is the first step of morphogenesis

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Gastrulation is the first step of morphogenesis
Morphogenesis is the process whereby individual
cells undergo complex movements that generate the
organ rudiments. Gastrulation generates the three
basic germ layers from which organs arise. How
do sheets of cells (epithelia) move during
gastrulation? 4 methods.
Invagination is the local inward movement of
cells from a cavity
Involution is similar, but more dramatic. It is
an inward expansion of epithelial cells around an
edge such as the blastpore.
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Convergent extension is elongation of an
epithelium in one direction while it shortens in
the other direction (stretching taffy). The cells
can keep their relative positions and elongate or
they can interdigitate.
Epiboly is spreading movement of an epithelium to
a deeper or thinner layer.
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How do individual cells move during gastrulation?
4 basic types of cell movement lead to the
changes in epithelial sheets that characterize
gastrulation. 1. Migration is the movement of an
individual cell over other cells or a substrate.
2. Intercalation is wedging of cells between
their neighbors. Lateral intercalation involves
lateral movements of cells in the same layer
between one another convergent extension.
Radial intercalation involves wedging of 2
different layers. This process often leads to
epiboly, the surface area of the epithelium
increases while the thickness decreases.
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3. Ingression is the movement of individual cells
from an epithelium into an embryonic cavity.
4. Shape changes are coordinated changes in cell
shape that cause an epithelium to invaginate,
buckle or undergo convergent extension.
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Embryonic cells are broadly classed as epithelial
or mesechymal
Epithelial cells are well-differentiated. They
compose skin and line the body cavities (ie, the
digestive tract). They are polarized. Their
apical surface faces out and their basal surface
rests on the basement membrane (extracellular
matrix that supports cells). Epithelial cells are
closely connected with adjacent cells by
specialized attachments including tight
junctions, gap junctions, and desmosomes.
Mesenchymal cells are poorly differentiated and
have the potential to develop into many different
tissues, including epithelial cells. They have a
leading edge with lamellipodia, and a trailing
edge. They are not connected to adjacent cells
but they are in contact with the extracellular
matrix.
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Gastrulation in sea urchins
Primary mesenchymal cells these cells change
adhesive properties and the large micromeres
start to migrate into the blastocoel as free
mesenchymal cells (ingression). Mesenchymal cells
are loose cells that can differentiate into many
different organs. Archenteron the primitive
gut. The archenteron is formed in several stages.
1. the vegetal plate invaginates into the
blastocoel, 2. It elongates by convergent
extension. 3. It hooks up with the front and is
pulled forward, and 4. Involution occurs with
movement of cells around the blastopore and into
the archenteron. What forces drive the process
of gastrulation?
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The primitive groove and pit are the site of
gastrulation in birds
Crossection of blastoderm (blastula in
birds). Epiblast is the upper layer of epithelial
cells, blastocoel is the space below the
epiblast, and hypoblast is the lower layer of
epithelial cells.
Epiblast cells roll over the primitive ridge and
involute into the groove. The cells lose contact
with one another and migrate inwards by
ingression Mesoderm. 3 germ layers are
established
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Gastrulation in a 16 day old human embryo

• The primitive streak and Hensons node form just
  as in birds.
• The cell movements are similar
• Cells roll over the primitive ridge and into the
  groove
• They ingress individually and move out to form
  discs with the 3 germ layers
• The 3 layers also move laterally to form the
  extra embryonic endoderm and
• mesoderm even though there is no yolk to
  digest. This is surprising
• because mammalian embryos could gastrulate
  easily by invagination as sea
• urchins (they have a placenta and no yolk).
• Instead, gastrulation appears to recapitulate a
  pattern established by bird-
• like ancestors and reptiles.

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Molecular control of gastrulation and
morphogenesis
Does each cell in the blastula have detailed
instructions in the DNA that tell it exactly
where to go during gastrulation?
If an embryo is disaggregated into individual
cells, each should know exactly where to go to
reform a new embryo. When this experiment is
performed, a degree of reorganization occurs, but
it is not complete. Embryoids are slightly
similar to embryos but they lack the real
organization. Conclusions 1. Genes impart only
partial instructions for assembly of the
embryo 2. Like cells all stick together,
revealing distinct adhesive properties. 3. The
relative positions of aggregates reflect the
relative positions in the embryo (skin outside,
heart inside).
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Cell adhesion is the driving force in gastrulation
When cells from an embryo are disaggregated and
recombined, they can be readily ranked according
to their ability to form the central
portion. (chondrocytes gt heart cells gt liver
cells is the hierarchical order)
Differential adhesion hypothesis the cell type
with maximal adhesiveness (chondrocytes) will
form a core that is surrounded by concentric
spheres of cells with progressively lower
adhesiveness. Cell adhesion can be measured by
the pancake test. When aggregates of different
cell types are subjected to a flattening force
(centrifugation to induce a centrifugal force),
the cells that adhere most tightly form a ball,
while those that adhere more loosely form a
flatter, pancake structure. Cell adhesion is a
major factor that regulates aggregation of like
cells and controls position during morphogenesis.
What regulates how tightly or loosely cells
attach?
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Cells adhere by cell junctions, cell adhesion
molecules, or substrate adhesion molecules
Cell junctions large, complex structures that
form slowly but generate very strong and durable
connections (tight junctions, desmosomes, and gap
junctions). Cell adhesion molecules (CAMs)
single molecules that traverse cell membranes and
allow cells to adhere to one another. Adhesions
form quickly, they are selective, but they are
relatively weak in comparison to cell
junctions. Substrate adhesion molecules (SAMs)
a group that consists of extracellular matrix
molecules and matched receptors that are
expressed on the cell surface.
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CAMs firmly anchor adjacent cells to the
cytoskeleton
Cell adhesion molecules (CAMs) are glycoproteins
with 3 major domains The extracellular domain
allows one CAM to bind to another on an adjacent
cell. The binding can be to the same type of cell
(homotypic) or to a different cell type
(heterotypic). The transmembrane domain links
the CAM to the plasma membrane through
hydrophobic forces. The cytoplasmic domain is
directly connected to the cytoskeleton by linker
proteins. This anchoring is important to prevent
lateral diffusion of adhesion molecules in the
membrane.
Three major types of CAMs are immuno
globulin-like CAMs, cadherins, and lectins.
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Neural cell adhesion molecule is typical of
immunoglobulin (IgG)-like CAMs
N-CAM was one of the first to be discovered. The
extracellular domain has IgG like repeats that
are thought to allow binding to other N-CAMs by
interdigitation between loops. Insects have IgG
CAMs but no IgG. Thus, IgGs may have evolved from
IgG-like CAMs.
Polysialic acid region (PSA) 3 long carbohydrate
chains with negative charge are attached to the
5th loop. The overall charge varies on different
N-CAMs. Large PSA regions induce a large negative
charge which repels cells (embryonic cells during
gastrulation). Small PSA regions allow attachment
due to low charges, and these are common on adult
cells.
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Cadherins mediate calcium-dependent cell adhesion
Cadherins are the most prevalent CAMs in
vertebrates. They are rapidly degraded by
proteases in the absence of Ca. There are 4
major types
E cadherins in epithelial cells P cadherins in
placenta N cadherins in neural tissue L cadherins
in liver Each associates with its own type.
125 kD transmembrane glycoproteins that bind
homotypically using the first 113 AA The
differences in cadherin expression are
responsible for the differential adhesiveness
seen in disaggregated tissue. Cells that express
more cadherin tissues that form a ball in the
center of cell aggregates.
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Integrins mediate adhesion to ECM
Integrins are a family of transmembrane
glycoproteins that are composed of 2 chains, a
and b. There are 40 different types of a chains
and 8 types of b chains that can combine to form
a large number of different integrin
molecules. The a chain has binding sites for
Ca and Mg which are needed for integrins to
adhere. The 2 subunits form the site that binds
to the RGD domain on ECM. The cytoplasmic tail
of integrins is connected to a linker protein
that connects to the cytoskeleton. A bridge from
ECM to cytoskeleton.
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CAM expression during gastrulation is correlated
with cell fate
Fate map it is possible to predict which parts
of the blastula will develop into specific
structures after gastrulation. Expression map of
CAMs it is possible to localize expression of
CAMs using in situ hybridization and
immunostaining of the blastula. Cells with
different fates express different CAMs. Cells
destined to become neural tissue express high
levels of N-CAM. Cells destined for epidermis
express E-cadherin.
The respective cell adhesion molecules are
expressed before the cells actively start to form
the adult tissue. This suggests that CAM
expression is important in fate
determination. During gastrulation, cells go
where their CAMs lead them
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Changes in cell adhesion are important for
gastrulation
Gastrulation in the sea urchin is initiated by
specific changes in cell adhesion. One of the
first steps is ingression of mesenchymal cells
from the vegetal plate into the blastocoel to
form the skeleton of spicules. The mesenchymal
cells lose their adhesion to hyaline and the
adjacent nonmesenchymal blastomeres. They start
to increase adhesion to the basement membrane and
material within the blastocoel. These changes can
be measured by isolating specific cells and
testing adhesion in culture. E-cadherin is lost
from the ingressing cells due to endocytosis of
specific areas where it was expressed. Levels of
b-catenin are also reduced on these cells.
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Fibronectin on the inner roof of the
blastocoel is critical for gastrulation
Immunostaining of the blastocoel shows that
fibronectin was expressed in abundance on the
inner roof. Fibronectin binds to integrins on the
membrane. Neutralizing antibody to fibronectin
was injected into the blastocoel to test the role
of fibronectin. This aborted gastrulation. Since
no epidermal cells could migrate into the
blastopore, many cells accumulated on the
surface, forming deep folds. If an unrelated
antibody was injected, there was no
inhibition. Fibronectin binds integrins through
an RGD sequence. Similar results were obtained by
injecting the tripeptide RGD. Furthermore,
blocking the integrin receptor with injected
antibodies also inhibited gastrulation.
Fibronectin is important for contact guidance of
migrating cells during gastrulation.
19
PC12 cells resemble chromaffin cells which can
differentiate into neurons. When they convert to
the neural phenotype they express N-CAM and
N-cadherin on their cell surface. When PC12
cells are grown on cells that do not express
N-CAM or N-cadherin (3T3 cells) they retain the
undifferentiated chromaffin phenotype.
If the PC12 cells are grown on the same cells
that have been transfected with N-CAM or
N-cadherin genes, they convert to the neuronal
phenotype. They form long dendrites and express
neuronal genes. Differentiation is accompanied
by opening of calcium channels
20
Organogenesis in humans is essentially complete
after 6-8 weeks
The 5 week old human embryo has a head with
rudiments of eyes, ears, and brain. It also has a
trunk with tail and limb buds. The structures
develop rapidly and the organs are basically
formed before 8 weeks. The period of
histogenesis, when cells acquire functional
specialization, then begins and continues
throughout development fetus.
Organogenesis involves many of the same cell
behaviors that occur during gastrulation.
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Neurulation is of scientific and medical interest
Spina bifida one of the most common birth
defects in humans. Due to defects in closure of
the neural tube and malformations of brain and
spinal cord. There are several forms that differ
in severity Spina bifida occulta the mildest
form is caused by failure of a vertebrate to fuse
dorsally. It causes no pain or neurological
disorder. It is very common and as many as 10
have this minor defect. The only sign of its
presence may be a dimple or a tuft of hair
normal
spina bifida occulta
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What forces cause closure of the neural tube?

• Closure of the neural tube is mediated by 3
• effects
• Apical constriction of neural plate cells
• Rapid anteroposterior extension
• Cell crawling
• 1. Apical constriction a band of
• microfilaments in the apical region of neural
• plate cells contracts and causes cells to
• assume a wedge shape. This causes the
• neural plate to bend and form the hinge
• regions.
• Apical constriction occurs in the midline
• throughout the neural plate. It also occurs in
• the lateral folds to form the mediolateral
• hinge sites.

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Spemanns famous organizer experiment
Spemann originally thought that the donor dorsal
lip of the blastpore differentiated into neural
tube and structures of the embryonic axis. To
prove this, he grafted dorsal lip blastopore
tissue from a nonpigmented donor newt onto
another gastrula from a heavily pigmented
species. When the embryos developed he found that
the embryonic axis was actually composed of
pigmented recipient tissue. Only a small strip
of donor tissue was present in the middle of the
neural plate.
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Spemanns experiments were confirmed in birds

• 3 conclusions about the organizer
• The dorsal lip of the blastopore developed
  according to its own fate. It gastrulated
  normally in the new location and formed
  notochord.
• The graft dorsalized the hosts ventral mesoderm
  (converted gut tissue to kidneys and somites).
• The graft acted as a neural inducer. It caused
  host ectoderm to form a neural plate and close to
  become a neural tube.

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Axis induction by disinhibition
Early experiments by Spemann studied how the
organizer worked. If the cells of the dorsal lip
were killed or crushed, the activity was still
present. Activity could be mimicked partially by
changes in pH or ionic strength.
Disinhibition the normal pattern for ectodermal
development is neurulation. Ventral ectoderm
escapes from this path by producing an inhibitor
of neurulation. The organizer works by inhibiting
the inhibitor. Cells cultured from ventral
ectoderm differentiate as neurons only if
cultures are sparse. Bone morhogenetic protein
(BMP-4) is released by ventral ectoderm cells. It
stimulates formation of ventral structures and
inhibits neurulation if injected into
embryos. If BMP-4 is blocked, dorsal structures
replace ventral structures (dominant negative
mutants or innactivate the BMP-4 receptor).
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Spemanns organizer (dorsal lip) inactivates BMP-4
Chordin and noggin the dorsal lip of the
blastopore produces 2 proteins that antagonize
the action of BMP-4. Chordin and noggin each bind
to BMP-4 and prevent it from binding to its
receptor. Chordin and noggin have strong
dorsalizing effects. They cause excessive head
development and block ventral differentiation if
injected into the embryo.
What turns on chordin and noggin? Goosecoid may
be the master regulator that is similar to
Spemanns organizer. Expression of goosecoid is
limited to the dorsal lip of the blastopore. It
is turned on at the right place at the right
time. Goosecoid encodes a transcription factor
that can activate chordin gene expression. Work
is continuing to explore how this gene functions.
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Histogenesis is the process by which cells and
tissues acquire functional specialization
embryo
fetus
Cleavage ? gastrulation ? organogenesis ?
histogenesis (2 weeks) (1 week)
(4 weeks) (7 months) Organogenesis
the formation of organ rudiments to establish the
basic body plan. Histogenesis differentiation
of cells within the organs to form specialized
tissues. Tissues are composed of cells and
extracellular material that perform a specific
function. Each specific tissue develops mainly
from one germ layer.
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The spinal cord develops a dorsoventral pattern
All nervous functions depend on development of
complex connections between neurons. These
circuits start to develop in the embryo. Alar
plate as neurons and glial cells accumulate in
the mantle, they form ridges on either side of
the neural tube (dorsal or afferent columns).
These will develop into afferent nerves that
conduct signals to the brain. Basal plate
accumulation of cells in the ventral region
produces basal or efferent columns. These will
develop into efferent neurons that carry signals
to muscles and organs (motor neurons). The gray
matter in the mantle layer is composed of cell
bodies of neurons and the white matter in the
marginal layer is composed of myelinated axons.
The floor and roof plates are composed of glial
cells. What causes this pattern?
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Sonic hedgehog (shh) induces the dorsoventral
pattern
Sonic hedgehog (shh) is a gene that is expressed
in the notochord at first and later in the floor
plate. Mice that lack shh fail to develop floor
plates in the CNS. Shh is a secreted
glycoprotein that induces a gradient that is high
near the floor plate and progressively lower in
dorsal regions. Shh initially induces neural
plate cells to form floor plate. Other signals
from the dorsal ectoderm direct the dorsal
columns and the roof plate. Different levels of
shh appear to specify different types of neuron
differentiation.
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Different concentrations of shh induce distinct
types of neurons
In the developing spinal cord, the floor plate
produces shh and creates a concentration
gradient. Motor neurons develop closest to the
floor plate, type 2 interneurons are next,
followed by type 1 interneurons. To see whether
the shh gradient is really important, isolated
cells from neural tubes were cultured in various
concentrations of shh. Cells were stained with
antibody specific for floor plate, motor neurons,
or type 1 or 2 interneurons.
Bone morphogenetic protein released by the dorsal
ectoderm and roof plate has an analogous function
in generating the dorsal columns
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Neural crest cells form a variety of tissues
The fate of neural crest cells has been mapped by
a number of techniques (radioactive tracers,
transplants from pigmented species to albinos).
There are 2 patterns of migration in the trunk
region Dorsolateral path enter skin and form
melanocytes Ventral path form afferent neurons
of dorsal route ganglia, sympathetic and
parasympathetic ganglia, and adrenal medulla
Neural crest cells help to form addition
structures in the head such as bones, connective
tissue, eyes, ears, and teeth. They also help
to form blood vessels and connective tissue in
the trunk
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How do neural crest cells differentiate into many
tissues?
Pluripotency hypothesis each neural crest cell
has the potential to form any or all structures.
Inductive signals from adjacent tissue determines
their fate. Selection hypothesis the neural
crest contains a mixed population of
predetermined cells. Each cell has only one
possible fate and it migrates according to this
fate.
The real truth may lie between these two
extremes. Clonal analysis when individual
neural crest cells are placed in culture, it is
clear that a single cell can give rise to others
that differentiate into multiple cell types
(pigment cells and neurons). Premigratory cells
have a wider potential than do the cells that
have already started to migrate. They may become
partially differentiated as they migrate.
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What are the molecular signals that control
differentiation of neural crest cells?
Extracellular matrix (ECM) neural crest cells
constantly extend filopodia to feel the
ECM. Pieces of filter were placed in an embryo
at the dorsolateral or ventral pathways. After
the filters absorbed ECM, they were removed to a
culture dish and allowed to interact with neural
crest cells. Dorsolateral ECM induced
melanocytes and yellow pigment cells. Ventral ECM
induced neurons. No matrix allowed the cells to
remain undifferentiated.
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The otic placode forms the inner ear
In humans, the otic placode appears by the third
week on both sides of the rhombencephalon. The
otic vesicle is induced by the underlying neural
tissue in the rhombencephalon. It invaginates to
form the otic pit and then pinches off to form
the otic vesicle. Ganglion cells develop from its
medial surface. Labyrinththe otic vesicle
expands unequally and constricts in other areas
to form a complex shape. The cochlea develops to
sense sound and the semicircular canals form to
serve as an organ for balance and body position.
36
Formation of the eye involves reciprocal
interactions
Lens placode the ectoderm invaginates in
response to signals from the optic cup
underneath. It then pinches off as a lens
vesicle. Cells elongate to fill the vesicle and
start to synthesize crystallins. Optic cup
forms from the neural tube by invagination. The
opening (choroid fissure) closes forming a round
optic cup, an extension of the brain. Optic
stalk connection to the brain that is filled
with neurons to form the optic nerve. Reciprocal
interaction the lens induces the formation of
the optic cup and the cup regulates formation of
the lens. When the lens from a species with large
eyes is transplanted, it induces an extra large
optic cup and it also does not grow as large as
usual.
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Nasal placodes form the olfactory epithelium
In humans, the nasal placodes appear after the
4th week. They are induced by the underlying
telecephalon. During the fifth week, nasal
swellings appear around the placodes which now
become the two nasal pits. The pits start out
far apart, but the 2 maxillary swellings grow
large and push the pits to the center. The medial
parts of the nasal swellings fuse to form part of
the upper lip. Olfactory epithelium the
original lining of the nasal pit comes to rest on
the roof of the nasal cavity. It forms the
epithelium that senses smell and connects to
neurons in the telencephalon. The nasal cavity
becomes continuous with the pharynx.
What can go wrong?
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How does skin develop and differentiate?
Epidermis the largest derivative of ectoderm
forms the outer layer of the skin. It is an
epithelium and cells are connected by desmosomes
and tight junctions. The epidermis consists
initially of two layers periderm is a temporary
outer layer and the germanative layer lies below.
The germanative layer is composed of stem cells
that divide actively to produce differentiated
progeny. The basal layer develops from the
germanative layer (it contains stem cells in the
adult).
Spinous layer forms as cells are squeezed out of
the basal layer. They become large and
differentiate. Granular layer starts making
keratin granules Cornified or horny layer is
composed of dead cells that are filled with
keratin It takes cells about 7 days for each
cell to journey through the skin
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Hair development in humans
The underlying mesenchyme in skin forms a dermis,
a layer of connective tissue just below the
epidermis. The dermis induces a variety of
epidermal structures depending on the species
(feathers, hair, scales).
Hair bud hair formation begins as a small bud
that that penetrates the dermis. It is induced by
a group of mesenchymal cells and the hair bud
then envelopes these cells to form a hair
papilla. Hair follicle is the entire
organ Sebaceous glands are induced to form on
the side. The hair shaft is formed when the
inner cells of the follicle start to
differentiate and produce keratin in the form of
hair. The continued production of keratin by the
cells at the base of the shaft causes the hair to
grow longer.
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Mammary gland development in humans mimicks
ancestral patterns
In normal development of humans, only one pair of
segments in the ridge survives, and the remaining
precursors degenerate. In some individuals, the
other segments of the mammary ridge fail to
degenerate, so that accessory nipples or breasts
are formed.
Primates evolved from small creatures that nursed
multiple offspring. An extended mammary ridge
would have given human ancestors a survival
advantage. Two breasts are obviously more
adaptive for humans who normally have only one
offspring at a time. Atavism the occasional and
abnormal persistance of a primitive adult feature
in an evolved species (multiple mammary glands).
It is easier to modify an older pattern of
development than to develop a totally new
pattern. This idea is a pervasive in
developmental biology.