Chapter 11 Organogenesis
The vertebrate limb and tooth development
Organs are formed due to interaction between different tissues.
Model systems used for limb development are for example the mouse. By looking at
spontaneous mutations and gene-knock-outs, you can discover which genes are responsible
for limb formation. Chicken can also be used because it is accessible for microsurgery. The
pectoral fin of the zebrafish is often used as a model system as well.
Chicken limb
The wing bud and leg bud form the limbs. There are multiple axes in the limb bud, proximal-
distal, dorsal-ventral and anterior-posterior. The wing of the chicken only has 3 digits, these
are called 2,3 and 4. → There are 3 axes and 3 digits.
When the chick has developed after 10 days you have formation of cartilage which
will be replaced by bones. After 10 days, the feather buds and muscles and tendons are
formed as well. When talking about fore limbs and hind limbs, the stylopod is the most
proximal part of the limb with Humerus in the forelimb and Femur in the hindlimb.
Zeugopod is the next part containing the radius, ulna, tibia and fibula. Most distal is the
Autopod region with carpal bones and tarsal bones, metacarpals, metatarsals and
phalanges. →The limbs consists of stylopod – zeugopod – autopod from proximal to distal.
Limb buds are composed of mesoderm with an outer covering of ectodermal cells
which form the skin and the mesenchymal cells form lateral plate mesoderm and somites
from migrating muscle cells. Along the tip of each limb bud runs a thickened ridge of
ectoderm, the apical ectodermal ridge (AER).
Axis determination in limb development PD-axis
P-D = proximal distal axis. This axis contains the Apical Ectodermal Ridge (AER).
The limb bud will grow out of the AER, this is a boundary between the dorsal and ventral
limb bud. Cells behind AER differentiate from proximal to distal.
Chick will end up with 3 digits and the mouse with 5 and time of development is different
but looks similar. Cartilage develops from proximal to distal and later it is replaced by bone.
There is a dip and the elbow will form there. The hand-plate or digital plate is where the
digits will develop.
The limb bud knows how to develop at the right positions due to HOX-genes. They
regionalize the body, the HOX genes determine that a certain part has to become the limb
bud. HOX-genes come regionalized in lateral plate mesoderm. The combination of HOX-
genes controls Tbx5 (forelimb region at 3’ end) and Tbx4 (hind limb at 5’ end). Pitx1 is also
expressed in the hind limb region and is required for Tbx4 expression and specifies the
identity of the hindlimb. Tbx5 and Tbx4 both control FGF10 expression and this induces
expression of FGF8 in the overlying ectoderm what will form and maintain the apical
ectodermal ridge and the polarizing region with Sonic Hedgehog expression.
1
,AER is required for proximo-distal development. If you remove the AER, proliferation of limb
bud is reduced, and cell death occurs. The proximo-distal outgrowth depends on the time at
which the wing is removed. The later the AER is removed, the more limb will already be
developed. Limbs develop in proximo-distal sequence, proximal is determined earlier.
What signaling molecules are responsible for P/D-patterning? → FGF.
Signaling what is responsible for the limb growth: FGF. When you remove the AER and have
beads soaked in FGF placed at the tip, a normal wing can develop. One type of molecule can
instruct a whole organ to develop. The limb gets a long shape and not a broad shape, this is
due to oriented cell movement and cell divisions. Before limb bud formation, cells divide in
head to tail elongation, they are oriented parallel to the antero-posterior axis of the body.
The early limb bud forms as a result of the anterior cells of the LPM (lateral plate mesoderm)
moving posteriorly and lateral cells move perpendicular to the body axis into the direction of
the budding. After the limb bud region is determined, cells recruited from lateral plate
epithelium undergo epithelial-to-mesenchymal transition. The distal cells move faster than
proximal cells. In the elongating limb bud, cells in central region are oriented along PD-axis.
Cells in the dorsal and ventral region are oriented towards ectoderm. FGF and Wnt signaling
pathways play a role in this process. When Wnt5a is mutated cells don’t go in a specific
direction, but they move randomly. It is a molecule that retracts cells and instructs them to
migrate towards their source. The Wnt/Planar cell polarity pathway is a non-canonical
pathway. Signaling by Wnt ligand through receptor leads to changes in the cytoskeleton.
Wnt5a acts a chemoattractant. Wnt5a expression is highest at the limb bud tip (AER) and
expressed in all regions of the embryo that undergo growth.
2
,Proximo-distal patterning
Cell have to acquir a certain identity. There are two models: 1) Progress zone model, 2) Two
signal model. Cells acquire positional values along the axis. The main signaling regions are
the AER (acquired for P/D patterning) and the polarizing region in the posterior (acquired
for A/P patterning).
Progress zone model
Cells acquire positional information, and cells interpret information according to their
developmental history. Cells in the zone of proliferating undifferentiated cells remember
how long they are at their place and will know when to differentiate. The time spent in the
progress zone determines the structure the cells from, the earlier cells leaving the zone will
from different structures than cells leaving later. FGFs maintain the progress zone, cell
measure the time they are exposed to FGF.
Two-signal model
Cells in the early limb bud acquire positional values from the presence of two opposing
signals: one due to a source of retinoic acid (RA), in the most proximal region, which
specifies proximal, the other due to FGF from the AER which specifies the distal. As the limb
bud grows, cells that are out of range of both signaling sources (in the middle), acquire and
intermediate positional value. As the limb grows, the different domains expand, with the
proximal domain expanding first and then successively more distal domains. → Opposing
signals specify cell fate: FGF and RA.
3
, Polarizing region or Zone Polarizing Activity (ZPA)
Polarizing region/zone of proliferating cells. When
you transplant this part into the anterior you get
full duplications. When the polarizing region of an
early embryo is grafted into the anterior margin of
another chicken wing bud, a mirror image pattern
is created. Smaller grafts or a shorter time leads to
a smaller duplicated region, for example only digit
2.(afb 14)
Anterior posterior axis is patterned by a
morphogen of Shh. It is present in the posterior
limb bud and has a graded protein distribution.
You can induce a leg by implanting FGF4 in the
flank (ectopic), it grows a limb bud and also expresses Shh. But now it is present in the
anterior and not posterior. Now the digits are formed 4,3 and 2 they will from the most
posterior digit first. → FGF is important in establishing in the PD-axis and AP-axis.
(Discussed in Chapter 8)
FGF8, HOXD and HAND2 regulate Shh and signaling by Hh leads to active GLI3/Ci, this
activates target genes in the nucleus. → The ratio GLI activator to GLI repressor in a cell
reflects Shh signaling. Because Sonic Hedgehog acts as a morphogen, its spatial
concentration in tissues varies and cells respond differently at different threshold
concentrations. There is diffusion of Shh through tissue. Morphogens are used
regionalization of the AP and DV-axis, vertebrate patterning and patterning DV-axis along
the neural tube.
4
