Embryology 1 :
The Genetics of AnteriorPosterior Axis
Determination
Major Axes in Body
Plan Development
 Embryos develop three axes: the anteriorposterior axis (from head to tail), the
dorsal-ventral axis (from back to belly)
and the right-left axis
 Structures show a specific localization
(neural tube is a dorsal structure in the
embryo and the heart is on the left side of
an adult body)
 The specification of axes occurs early
during embryonic development
Major Axes in Body
Plan Development
 The specification of early embryonic
cells is due to cytoplasmic determinants
stored in the oocyte
 The cell membranes that form during
cleavage establish the region of
cytoplasmic determinants that direct
different gene expression in every
blastomere
Drosophila as a Model for
Molecular Embryology
 Drosophila is easy to breed, cheap to rear
in the lab
 It is hardy and prolific (genetic studies are
possible)
 A genome-wide approach has identified
the molecules for body plan development
A/P Polarity in the Adult
■ The body comprises three thoracic and eight abdominal segments
■ Every segment is specific and has different appendages: T1 has
only legs, T2 legs and wings, T3 legs and halteres
■ The body is organized accordingly to a “plan”
Drosophila Early Embryonic
Development
■ The zygotic nucleus divides several times without formation of cell membranes
■ The specification of the A/P and D/V axes is accomplished by interaction of
molecules in the same single multinucleated cell
The Question
How do we get from an homogenous
egg to a body that is a collection
of different segments?
General Principles
 The determination of the A/P axis is due
to a cascade of interacting genes
 These genes act sequentially to first
divide the embryo in several segments
 Segments acquire then their specific
identity
Cascade of Genes Determining A/P Axis
Maternal genes
Hunchback
Gap genes
Pair-rule
Segment polarity
Homeotic genes
Bicoid and Hunchback at the Anterior Tip
Ovary
Embryo
Bicoid mRNA
Nurse Cells
Oocyte
Bicoid Protein
A
P
In the mother ovary the bicoid mRNA produced by accessory cells, the nurse
cells, is deposited in the oocyte and it is tethered to the anterior tip by
microtubules
The bicoid mRNA is translated into the corresponding protein which forms a
gradient with the highest concentration at the anterior tip
The same gradient is also created for hunchback, another anterior determinant
Nanos and Caudal at the Posterior End
Nanos protein
A
P
Similarly, nanos mRNA is given to the egg by the mother and it is bound to
the posterior region of the unfertilized egg by interaction with the
cytoskeleton
A gradient for the corresponding protein is created as reported in the picture,
with the highest concentration at the posterior end
A similar gradient is also created for Caudal
Early A/P Determinants
Four gradients are present: an A/P gradient for Bicoid and Hunchback and
a P/A gradient for Nanos a Caudal
All these proteins function as transcription factors that can activate the
expression of the following genes in the cascade, the gap genes
Summary
 Positional information has been
generated: the presence of Bicoid and
Hunchback “label” the anterior end,
Nanos and Caudal the posterior one
 Embryos defective (mutant) for Bicoid
lack the anterior structures while
embryos mutant for Nanos lack the
posterior part
THE GAP GENES
giant
Kruppel
Knirps
■The Gap Genes are expressed into broad regions of the embryo
■ Mutations in the gap genes produce embryos lacking a series of
contiguous segments corresponding to the segments where the gap gene is
expressed
The Question
How do we get from an embryo with smooth
gradient of proteins to an embryo
characterized by proteins expressed in
broad stripes?
Regulation of Gap Gene Expression
I
II
Stripe I requires high
level of Hb protein
Stripe II requires
high level of Caudal
Kr requires low levels of Hb
II
Stripe II requires low
levels of Caudal
The Pair-Rule Genes
Even-skipped
Stripe 2
The pair rule genes are expressed in a zebra-like pattern dividing the
embryo in 15 sub-regions where a vertical band of nuclei express
the gene and the next one does not
The stripe 2 of eve is repressed by both high levels of giant and
Kruppel
Regulation of eve stripe 2
The expression of eve stripe II is limited to the “valley” region between
high levels of giant and Kruppel proteins since both are repressors
Bicoid and Hunchback activates the expression of eve in the same stripe
We are half-the way done!
Drosophila Early Embryonic
Development
■ The zygotic nucleus divides several times without formation of cell membranes
■ The specification of the A/P and D/V axes is accomplished by interaction of
molecules in the same single multinucleated cell
The Segment Polarity Genes
■ The segment polarity genes reinforce and maintain the periodicity generated
by the pair-rule genes
■ They are expressed in 14 stripes along the A/P axis
■ Their expression is regulated by the pair-rule genes
Regulation of Segm-Polarity by Eve, Ftz
Fushi-tarazu (Ftz) and even-skipped (eve) are expressed in close, non
overlapping regions
High levels of Ftz and Eve induce the expression of engrailed (en), a Segmentpolarity gene expressed in 14 stripes
Conversely Eve and Ftz inhibit wingless (wg). The expression of wg will be
confined in stripes running between the expression of Ftz and Eve where there
is no Ftz or Eve
Maintenance of en and wg Expression
by Reciprocal Interaction
Wg is a secreted protein. It binds to wg
receptor on the adjacent en cells and it
activates en.
En is a transcription factor and it
activates the expression of hedgehog
(Hh).
Hh is a secreted protein that binds to its
own receptor on wg expressing cells and
it activates the expression of wg.
En and wg establish a polarity in every
segment
Homeotic genes
Colinearity between the position of the gene on the chromosome and the
sequence of the segment they specify: Scr specifies T1 and precedes on the
chromosome Antp that specifies T2
Every homeotic gene represses the expression of the previous gene in the
segment they specify. Ubx identifies T3 and represses Antp in T3
When Something Goes Wrong…
Normal fly head
In Ubx mutants T3 becomes T2
Extra legs
Ectopic
expression of
Antp in the head
induces the
formation of legs
attached to the
head
Mammals and Drosophila
Every homeotic gene in Drosophila has its
homolog in mammals (Scr corresponds to
a5, b5 and c5 but the three genes in
mammals have the same function as Scr in
Drosophila)
Colinearity is also seen in mammals (panel
B)
When Something Goes Wrong…
Thoracic vertebra
Extra rib
Lumbar vertebra
The function of Homeotic genes in mammals is the same as in flies: the KO of
Hoxc8 in mouse causes an homeotic transformation: the first lumbar vertebra
becomes a rib. A rib is associated with the thoracic vertebra anterior to it
Retinoic acid as a teratogen
 RA can become a teratogen if present in
large amounts or at particular times
during development
 It is a secreted molecule involved in A/P
axis formation in mammals and in forming
the jaws
 It affects Hox gene expression in A/P axis
determination and it inhibits neural crest
cell migration from the cranial region of
the neural tube
The role of cell-adhesion
molecules in creating boundaries
between tissues
Cells do not sort randomly
Cells from the neural plate and from the epidermis were dissociated in
alkaline solution. When cells were mixed together, they re-aggregated and
they became spatially segregated: the neural cells are inside while the
epidermal cells stay at the periphery
How boundaries between tissues
are established
 Boundaries between tissues can be
created by different cell types having both
different types and different amounts of
cell-adhesion molecules
 The most common cell-adhesion
molecules are cadherins
 Cadherins establish and maintain
intercellular connections and they are
crucial in the spatial segregation of cell
types
Cadherins
Cadherins are Ca-dependent adhesion
molecules
They are anchored to the cell by a complex of
proteins called catenins
Catenins interact with actin cytoskeleton
They have an adhesive recognition site to
bind to similar cadherins
Cadherins join cells together by binding to
the same type of cadherins on another cell
(homophilic interaction)
Cadherins
Cells with E-cadherins (Epidermal cells) stick together and they will sort out
from cells containing N-cadherins (Neural Cells) in their membranes
Cadherins during gastrulation
During gastrulation the presumptive neural tube expresses N-cadherins while
the presumptive epidermis expresses E-cadherins
These tissues separate: the cells expressing N-cadherins invaginate to form
the neural tube while the cells expressing E-cadherins will form the epidermis
If the epidermis is experimentally manipulated to inactivate the E-cadherins
the cells will not hold together. If the N-cadherins are inactivated in the cells of
the presumptive neural tube, the neural tube will not form
The Extracellular Matrix as a
Source of Developmental Signals
 EM consists of macromolecules secreted
by cells into their immediate environment
 Cell adhesion and cell migration is
mediated by the EM
 EM is made up of proteoglycans, collagen
and specialized glycoproteins such as
fibronectin and laminin
Fibronectin
 FN functions as an adhesion molecule
linking cells to one another or to other
molecules (collagen and proteoglycans)
 FN has an important role in cell migration
 The roads over which migrating cells
travel are paved with FN
 FN leads germs cells to the gonads and
heart cells to the midline of the embryo
 Cells expressing integrins can bind
adhesion molecules
INTEGRINS
On the extracellular side integrins
bind to the sequence Arg-Gly-Asp
found in adhesion molecules
including fibronectins
On the intracellular side they bind
Vinculin and a-Actinin, these
proteins bind to Actin filaments
This dual binding allow cells to
move by contracting Actin
filaments against the EM