MCD BIO 138 Chapter Notes - Chapter lec 17: Lateral Plate Mesoderm, Limb Bud, Hox Gene
13 Oct 2020
School
Course
Professor
LECTURE 17: DEVELOPMENT OF THE VERTEBRATE LIMB
1. OVERVIEW
The development of the vertebrate limb requires the establishment of anterior-
posterior, dorsal-ventral and proximal-distal axes in the limb buds of the embryo. Cells
must differentiate to form different bones according to their position along these axes.
Position along each of these axes is controlled by secreted growth factors, and these
factors interact with each other so that the establishment of each axis is interconnected
with the others. The shapes and sizes of individual skeletal elements (bones) are
determined by the location of cells within the limb bud. In addition to secreted factors
that specify position along the axes, Hox genes provide AP axis information within the
limb and positional information that determines where limbs will form in the embryo.
We will discuss the mechanisms that lead to formation of limbs at discrete
locations along the A/P axis, and the mechanisms that specify cell fate within the limb
along the A/P and proximal-distal axes.
The A/P axis refers to the axis that runs from the thumb to the smallest digit. If
you hold your arms out away from your body, you can see that the A/P axis of your limb
runs in the same orientation as the A/P axis of your trunk.
The proximal-distal (P/D) axis refers (for your forelimb) to the shoulder to the tip
of our fingers.
The D/V axis refers (for your forelimb) to the side of the hand that has fingernails
(dorsal) and the palm (ventral). Holding your arms out away from your body
demonstrates that this is the same as the overall D/V axis of your trunk. We will not be
considering the specification of this axis.
2.LIMB EVOLUTION
The forelimbs and hindlimbs of tetrapod vertebrates share a highly conserved
basic pattern, even though forelimbs can look very different from the hindlimbs within a
species. There are also large differences in limb shapes between species, yet the basic
mechanisms of limb formation are highly conserved. For this reason, studying the
mechanisms that drive changes in the shapes of limb skeletal elements is a very
powerful approach to understanding how specific alterations in genes might lead to
changes in the body plan during evolution. The limbs of the first land vertebrates evolved
from the pelvic and pectoral fins of fish-like ancestors. The fossil record suggests that
the transition occurred when lobe-finned fishes living in shallow water moved onto the
land. The fins of these lobe-finned fishes contain bones that are directly analogous to the
humerus, radius, and ulna of the terrestrial vertebrate limb. These similarities cannot be
easily explained by convergent evolution, and thus are most likely due to common
descent.
More recent molecular studies have compared expression of signaling
molecules, discussed below, in fins and limbs. Many of the most important features of
the expression of these molecules are conserved. For example, Shh, discussed below,
is expressed at the base of the limb bud on the posterior side of both fins and limbs.
Similarly, Hox genes are expressed in a graded pattern in fins and limbs.
Examples of how changes in expression of Shh and its receptor have driven limb
evolution are discussed below.
3. FORMATION OF THE VERTEBRATE LIMB BUD
3.1. Specification of the Limb Fields
The first inductive stimulus for limb formation comes from the prospective limb
mesoderm, within the lateral plate mesoderm (recall from our discussion of gastrulation
in birds/mammals that this is the most ventral derivative of the mesoderm). Limbs form
as outgrowths from the lateral mesoderm at specific locations along the A/P axis. These
regions are controlled by the boundaries of expression of specific Hox genes. If lateral
plate mesoderm at an early stage is taken from a region where a limb would appear, and
is transplanted to a location where no limb would develop (e.g., flank, head), this graft is
able to induce limb development at these ectopic sites. The region of lateral plate
mesoderm that is competent to induce limb development is called the limb field. The
limb field also contains the information to specify the type of limb (upper limb, lower limb)
that is going to develop. This is under the control of the Hox genes. FGF10 is a signal
emanating from the lateral plate to the overlying ectoderm that induce limb development.
The Hox code is instrumental in determining where the limb field will form in the lateral
plate because the Hox genes expressed in the lateral mesoderm that is competent to
form a limb lead to induction of FGF10 specifically in these regions. FGF10 then induces
the initial outgrowth of the limb buds.
3.2 Signaling centers in the developing limb
In the limb field, on the flank of the embryo, mesenchyme from the lateral plate
mesoderm buds out and causes a protrusion of the overlying epidermis. This
mesenchyme, with its covering of epidermis, is referred to as the limb bud.
Experimental manipulations in the chick embryo, and morphological observations on
various vertebrate embryos, have led to the identification of three important components
of the limb bud: the zone of polarizing activity (ZPA) (also known as the polarizing
region), the apical ectodermal ridge (AER) and the progress zone (PZ).
We will see that the survival of each of these signaling centers is dependent on
the others. This enables coordination of limb growth along the proximal-distal axis with
outgrowth along the A/P axis.
3.3. Anterior/Posterior patterning: the polarizing region
The polarizing region, also commonly known as the zone of polarizing activity
(ZPA) is the posterior proximal region of the limb bud, and is the source of a polarizing
activity, or morphogen gradient. This is demonstrated by the fact that transplantation
of a polarizing region/ZPA to the anterior region of another limb bud results in a
duplication of the anterior-posterior axis of the limb. Thus, the identity and order of the
digits in the chick limb bud is changed from:
(anterior) II, III, IV (posterior)
to
(anterior) IV, III, II, II, III, IV (posterior).
We will discuss the identity of the signal produced by the ZPA that enables its activity,
and the evidence that this signal acts as a morphogen, in Section 4 below. Although the
above experiment demonstrates an essential role for the ZPA in A/P patterning, other
experiments demonstrate that the ZPA is also essential for proximal-distal
outgrowth. For example, removal of the ZPA leads to truncation of the limb due to a halt
in proximal-distal outgrowth. This is because the loss of the ZPA causes the loss of the
AER (discussed below).
3.4. Proximal-distal outgrowth: the AER.
The apical ectodermal ridge (AER) is a thickening of the epidermis at the dorsal-
ventral border of the distal edge of the limb bud. It displays a reciprocal interaction with
