Limb Development

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					ZO361- Lecture                                             Chapter 23, Section 23.5
                           Limb Development & Regeneration      Gilbert Chapter 16

I. General Limb Development
Outgrowth of embryonic body wall composed of a central core of loose mesenchyme
derived from the somatic part of the lateral plate mesoderm and the somites. This core of
loose mesenchyme is encased in an epithelial jacket derived from ectoderm. The
mesenchyme gives rise to cartilage or bone, muscle, and connective tissue.

Limb bud formation begins as an outgrowth of somatic mesoderm: increased cell
proliferation. The dermamyotome of somite migrates in at the same time to fuse with
somatic mesoderm to form a core of mesenchyme cells. These mesenchyme cells are
undifferentiated as they proliferate. Eventually these cells differentiate forming cartilage
first (derived from somatic mesoderm), then skeletal muscle (from myotomes of somite),
then connective tissue (somatic mesoderm) and dermis (dermatome of somite), which lie
below the epidermal (ectoderm) skin.

By day 3 (very early development of limb bud), the distal tip of limb bud (most outward
tip of limb bud) is covered with a ridge of thickened ectoderm called the apical epidermal
ridge (AER). This ridge is found in most vertebrates (not urodeles). The AER
disappears as the first digits form. The limb bud elongates by the continued proliferation
of undifferentiated mesenchyme at the tip of the limb (beneath the AER). During limb
extension, the more proximal cells (end toward the body) of the apical mesenchyme
proliferative zone show diminished mitotic activity. These proximate cells (fibroblasts
of mesenchyme) begin to differentiate. Therefore, differentiation of cells occurs in the
proximate to distal direction (body to tip). Thus, differentiation of limb elements occurs
in the temporal order of humerus, then radius/ulna, then wrist bones, then digits; under
control of the Hox genes (Figures 16.10, 16.14, and 16.15).

Interactions of the AER and mesenchyme proliferative zone for limb bud outgrowth (See
pages 628-630, Figure 15.4 and know the experiments demonstrating importance of AER
and mesenchyme interactions for limb outgrowth). Presence of AER is required for
continued proliferation of apical mesenchyme. If AER is removed, it does not regenerate
and mesoderm below ceases to divide, so limb development comes to a halt. If AERs are
grafted to side of limb, additional limb elements emerge under the 2nd AER
(differentiated cartilage forms 200 um proximal to the transplanted AER/proliferative
apical mesenchyme). The initial formation and continued existence of AER are
dependent on the underlying proliferative mesenchyme zone. If prospective limb
mesoderm is grafted to flank ectoderm, an AER forms and a supernumerary limb
develops. The mesoderm and AER produce as yet an unknown factor for maintaining
each other and limb outgrowth (See Figure 16.8).

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ZO361- Lecture                                             Chapter 23, Section 23.5
                           Limb Development & Regeneration      Gilbert Chapter 16
Key points to know:
    Mesenchyme induces the AER and specifies limb type
    AER sustains outgrowth and development of limb
    AER (present in the limb bud and disappears when digits form) keeps
      mesenchyme in a proliferative state, preventing cartilage formation
    FGF- regulates proximal to distal development
    Shh- regulates anterior to posterior development
    Wnt 7a- regulates dorsal to ventral development
    LPM induces myoblasts to migrate out from the somites and enter the limb bud.
      LPM cells become limb mesenchyme secreting FGF10. FGF10 secreted by the
      mesenchyme induces FGF8 in the AER, which signals back to proliferating cells.
    FGF induced limb at somite 21 and turns on Tbx5 and makes a wing.
    Ectopic Tbx4 in the flank causes FGF induced wing at somite 21 to form leg.
    Tbx4 appears to be critical for instructing hind limb development.
    Tbx5 is critical for forelimb development.

II. Establishment of Limb Axes
Three Axes: (See Figure 15.6)
   1. Proxidistal (P-D)- girdle to wing tip in chicks, shoulder to hand in humans
   2. Anterior-posterior (A-P)- digit II to IV in chick wing, thumb to pinky in humans
   3. Dorsoventral (D-V)- top of wing to bottom of chick wing, back of hand to palm in

By removing the region of mesoderm that will give rise to the limb (limb disk) and
transplanting to flank of host animals in a variety of orientations, investigators
established when and which limb axes are established first:

In amniotes (e.g. birds, humans)- A-P and D-V determined early on at around the same
time and P-D forms last. In salamanders and amniotes, P-D axes is established after
morphology of limb is apparent.

A-P and D-V Axis determination is generally a property of the mesoderm, not ectoderm.
However, in P-D axis, ectoderm may play a role also.

1. Establishment of Proximal-Distal Axes.

Progress zone - region of cell division under AER that allows limb to elongate from
proximal to distal.

Progress Zone Model: The fates of mesoderm cells within limb are determined by how
long they remain at the tip of limb (in the apical proliferative zone or progress zone).
Those that leave zone first turn into proximal structures (humerus). Those that leave later
will form more distal positional values & will form radius/ulna, then wrists, then digits.

Once mesoderm cells escape influence of AER and leave the progress zone, their fates
are irreversibly fixed, and the positional value (proximal distal position while limb is

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ZO361- Lecture                                              Chapter 23, Section 23.5
                            Limb Development & Regeneration      Gilbert Chapter 16
outgrowing) of a cell is determined the length of time it is under the control of AER (e.g.
those progress zone cells under influence of AER for the longest periods will form the
most distal structures).

Some regulation and the number of cell divisions may play a role in which cells form
distal or proximal structures.

2. Anterior-Posterior Axis

Specific by the Zone of Polarizing Activity (ZPA) a small block of mesoderm near the
posterior limb bud next to the body wall. Transplanting ZPA anteriorly causes mirror
image duplication (Figure 16.17).

Establishment of A-P Axes: Determined at the zone of polarizing activity (ZPA), which
is mesoderm that produces a factor called retinoic acid (derivative of vitamin A). ZPA
secretes retinoic acid, which diffuses along a posterior to anterior gradient (highest conc.
in the posterior axis, lowest on the anterior end). Retinoic acid mimics effect of ZPA.
Highest conc. of retinoic acid produces digit IV of chick (posterior end), medium
concentration digit III (middle structures) and lowest conc. digit II (anterior limb

The mechanism by which retinoic acid induces the A-P axes formation is via induction of
certain genes. Retinoic acid induces expression of sonic hedgehog gene whose protein
product activates changes in Hox A or Hox D homeobox genes along a gradient from
posterior to anterior end. Expression of homeobox Hoxa or Hoxd genes along a gradient
causes A-P limb patterning (Figure 16.14 and 16.15).

Retinoic Acid (RA) activates the HOX genes (HOX6) and limb bud initiation.
RA can also induce the tail stump to make limbs. Snakes do not have HOX6 gene,
therefore, limb formation is absent.

HOX genes specify the identity of a limb region. (Figure 16.4) HOX13 knockout mouse
lacks digits. HOXII knockout mouse lacks ulna and radius.
As the limb grows outward the HOX gene expression changes (Figure 16.5).

3. Dorsal-Ventral Axis

      Regulated by signals from the somites and lateral plate mesoderm
      Regulation by the ectoderm and responding dorsal mesoderm: Radical fridge
       (ectoderm), Lmx-1 (dorsal mesoderm)
      Wnt 7a expressed in dorsal ectoderm activates the synthesis of Lmx-1 in the
       dorsal mesenchyme. Mice mutant for the gene has soles and pads on both sides of
       the foot (no knuckle and nails). Wnt 7a maintains Shh expression in the ZPA and
       FGF4 in the posterior AER.
      FGF4 and Shh reciprocally maintain each other’s expression.
      Lmx-1 expressed in responding dorsal mesenchyme. Loss of function, LMX1
       have ventralized phenotype (nails on the bottom).

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ZO361- Lecture                                             Chapter 23, Section 23.5
                           Limb Development & Regeneration      Gilbert Chapter 16
      Engrailed is expressed in the ventral limb bud and limits the expression of radical
       fridge, Wnt-7a and Lmx-1 to the dorsal side.

4. The Three Axes are Coordinated
           1. FGF8 in the AER establishes the Shh expression in the ZPA
           2. Shh expression in the ZPA induces FGF4 in the AER
           3. AER and ZPA cross regulate to maintain FGF4 and Shh expression.
Experimental evidence: FGF8 may turn on Shh in the posterior cells made competent by
HOX8. Shh defines the ZPA. Implanting cells expressing Shh can cause mirror-image
duplication. Shh soaked beads can also have this effect.

5. Formation of Digits and Joints
See Figures 16.22, 16.23, and 16.24

          Programmed cell death
          Sculpts the limb. Interior necrotic zone separates the ulna and radius
          Interdigital necrotic zone separates the digits
          Digits and joints- involves cell death mediated by BMPs and Noggin

The Noggin gene blocks expression of the BMP gene in the organizer. Mice mutant for
Noggin fail to form joints as measured by GDF-5, a member of the BMP family. With
no Noggin to block it, excess BMP may enhance recruitment of cells into cartilage

BMPs induce mesenchymal cell to either undergo cell death or become cartilage-
producing chondrocytes. Both of these properties of BMPs are used in joint formation
under the regulation of Noggin.

III. Limb Regeneration
See sections 21.3 and 21.4, chapter 21 pages 534-540

Regeneration: the reactivation of development to restore missing tissues

Experimental Biology was born from a naturalist of the 18th century who wanted to
discover why some animals could regenerate, but humans cannot.

3 Major ways of Regenerating:
   1. Epimorphosis- Salamander Limb: involves de-differentiation of adult structures to
      form an undifferentiated mass of cells that then becomes re-specified.
   2. Compensatory Regeneration- Mammalian Liver: cells divide, but maintain their
      differentiated functions. Cells produce other cells similar to themselves and do not
      form a mass of undifferentiated tissue.
   3. Morphallaxis- Hydra: involves re-patterning of existing tissue and there is little
      new growth.

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ZO361- Lecture                                             Chapter 23, Section 23.5
                           Limb Development & Regeneration      Gilbert Chapter 16
•Epimorphosis- Salamander Forelimb
Involves dedifferentiation of adult structures to form an undifferentiated mass of cells
that then becomes respecified.

Formation of apical ectodermal cap and regeneration blastema

•Wound epidermis
- single layer structure that forms when limb amputation occurs
- first a plasma clot forms
- then epidermal cells from the remaining stump migrate to cover the wound surface

•Apical ectodermal cap
-proliferation region of the wound epidermis required for regeneration

•Regeneration blastema
- a proliferating mass of indistinguishable, dedifferentiated cells just beneath the apical
  ectodermal cap
- cells divide and redifferentiate to form the new structures of the limb
- cells lose the differentiated characteristics and become detached
- genes expressed in differentiated tissue are down regulated (example: myf5 muscle
- genes, which specify cell fate, are increased (example: msx proliferation gene expressed
   in progress zone mesenchyme)

See website 18.5 for polar coordinate model

Limb bud rotation experiment. When a newly emerged limb bud (or regeneration
blastema) is severed at its base and rotated 180 degrees on its stump, the result is a
limb with three areas of outgrowth. In this case, the limb bud proceeded to form a radius
and ulna with two new ulnas between them. At their tips were digits representing three
partial wings. (After Javois and Iten, 1986.)

Moreover, this patterning mechanism appears to be the same for both regenerating and
normally developing limbs. When axolotl limb buds were transferred to regenerating
axolotl blastema stumps, in a way that maintained the original polarity with respect to the
stump, normal limbs developed. However, when the polarity of the anterior-posterior axis
was reversed with respect to the stump, mirror-image supernumerary digits emerged
(Figure 2; Muneoka and Bryant, 1982). These results strongly suggest that the patterning
rules are the same for developing and regenerating limbs.

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ZO361- Lecture                                              Chapter 23, Section 23.5
                            Limb Development & Regeneration      Gilbert Chapter 16
See also Gilbert website 18.5 for affects
of retinoic acid on limb regeneration.
    - Anterior chick limb bud cells adjacent to a retinoic acid-soaked bead acquire
        ZPA-like properties. If the RA-exposed cells are grafted to the anterior margin of
        a host limb, they act as a ZPA and cause the formation of supernumerary digits.
    - Retinoic acid is seen as being capable of transforming anterior limb bud tissue
        into posterior limb bud tissue. The host limbs would now contain “posterior”
        tissue (the graft of retinoic acid-treated anterior cells) next to anterior tissue (the
        host limb bud). The result would be intercalary regeneration to restore the
        positions between the two normally non-apposed tissues. In this interpretation,
        retinoic acid is an agent that can modify positional values within the limb field.

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