## LIMB DEVELOPMENT AND REGENERATION

### LIMB DEVELOPMENT AND REGENERATION

11.          LIMB DEVELOPMENT AND REGENERATION

Embryonic development could be defined as the orderly, stereotyped process that adds complexity to the initial relative simplicity of a fertilized egg.

Human forelimbs and hindlimbs are derived from the pectoral and pelvic fins of primitive jawed vertebrates (gnathostomes) respectively. The first step in the development of a vertebrate limb is the determination of a group of embryonic cells that will give rise to the limb primordium (or limb bud). The areas of the foetus where limb buds are originated are known as a limb field. Limbs are present on the lateral side of the body.  Mesoderm layer present on the lateral side of the body is known as a lateral plate mesoderm. The limb fields are initially composed of cells within the lateral plate mesoderm (LPM) that are located in specific positions at both lateral sides of the embryo.

The development of vertebrate limb starts from limb field as a bud of mesenchymal cells that are induced by lateral plate mesoderm (LPM) and the growth of developing bud is regulated by a pseudostratified epithelium also known as an apical ectodermal ridge (AER). The determination of limb field in the trunk of the early embryo is specified by homeobox (Hox) or (T-box) Tbx genes; Tbx4 for hind limb and Tbx5 for forelimb, with the help of retinoic acid (RA) signalling. However, Tbx 2 and 3 is expressed in the area of forelimb and hind limb.

Limb induction in lateral plate mesoderm is mediated by the Fgf-8 gene, which encodes a member of the FGF superfamily of secreted factors. The Fgf-8 gene is expressed transiently and dynamically in the lateral plate mesoderm at the forelimb and hindlimb levels before and during limb induction, and the FGF-8 protein maintains cells in a proliferative condition. When FGF-8 protein is soaked in beads and grafted on the embryo, ectopic limb bud is developed. This indicates that the FGF-8 is capable of directing the initiation and normal development of an ectopic limb bud. The lateral side of the whole embryo is competent to form a limb.

11.1.      The (fibroblast growth factor [FGF-8])/FGF-10 Loop and Its Interaction with WNT Signals

Various model of limb development was proposed. But the limb initiation model is the best as the model indicated that localized sources of FGF-8 work as the signal to the adjacent lateral plate mesoderm cells to induce limb formation. Fgf-10 is also involved in the mediating the inductive effect of FGF-8 on the LPM cells. The Fgf-10 gene is initially widely expressed in the segmental plate (SP) and the lateral plate mesoderm. But around the stage 14 in the chick embryo, it becomes restricted to the LPM cells of the prospective forelimb area. FGF-8 controls the expression of the Wnt-2b gene in the LPM of the prospective forelimb area. Wnt-2b acts as an upstream regulator of Fgf-10 in the LPM.  That means FGF8 stimulates wnt 2b expression and wnt-2b stimulate fgf-10 expression. Thus fgf-10 expression is mediated by fgf-8. Thus mediating the induction of Fgf-10 by Fgf-8.

The expression of Wnt-2b  is stabilized in the somites, Intermediate Mesoderm (IM), and the LPM of the prospective forelimb area. The ectopic expression of wnt-2b alone can induce development of an ectopic limb in the later Fgf-10 expression is restricted to the prospective hindlimb area by wnt-8c. Both WNT-2B and WNT-8C proteins signal transduction involve $\beta$-catenin signalling.

The canonical WNT/$\beta$-catenin pathway appears to be both necessary and sufficient for induction of both forelimbs and hindlimbs through the control of fibroblast growth factor-10. Thus the fibroblast growth factor-8/fibroblast growth factor-10 loop is mediated by WNT/b catenin. Limb initiation is totally under control of wnt. Wnt signalling controls limb initiation, and localized expression of fibroblast growth factor-10 in the lateral plate mesoderm appears to be the key factor for limb induction. Mutation in the fibroblast growth factor-10 gene in mice results in the absence of limbs.

Apart from Tbx4 other members of Hox gene family Hoxc4 and Hoxc5 are also expressed in forelimb only. In hind limb, Pitx-1 gene is expressed which work as a transcription factor and expressed an Otx related subclass of paired-type homeodomain protein.

The Apical Ectodermal Ridgh (AER) is induced by its underlying mesoderm via fibroblast growth factor-10 & fibroblast growth factor-7 and the population of these mesodermal cells is regulated by fibroblast growth factor-8 & fibroblast growth factor 4 from AER.

The position of fibroblast growth factor-10 expression is regulated by Wnt8c in the hind limb and Wnt2b in the forelimb. The fibroblast growth factor-10 is a crucial signal for interaction among ectoderm and mesoderm because when a bead of fibroblast growth factor-10 is placed under the flanked ectoderm, it gives rise to an extra limb. The AER acts as a major signalling centre that runs along the distal margin of the bud, the major roles of this signalling centre are

Enabling the linear growth of bud by maintaining the plasticity or proliferation of mesenchymal cells.

Maintaining the expression for anterior-posterior axis determinants.

Regulating the cellular differentiation by interacting with the axis specifying proteins.

11.2.      Proximal- Distal axis specification

The limb bud elongates to give a fully developed limb by the elongation of the mesenchymal cell underneath the AER.

11.3.      Proximal-distal axis of limb has three region

Stylopod includes Humorous in forelimb and femur in the hindlimb. Zeugopod includes radio Ulna in forelimb and tibia-fibula in hindlimb and autopod includes digits. The structures along the Proximal-distal axis is determined by the expression of Hox genes. Hox genes give the signal to the mesenchymal cell which differentiates either stylopod, zeugopod or autopod.

Hox genes were first identified in Drosophila melanogaster. Hox gene encodes homeodomain transcription factors shown to provide spatial cues during the development of many embryonic structures in vertebrates and invertebrates including those that allocate the limb fields in a variety of organisms. Both in Drosophila and vertebrates such as mouse and chick, specific combinations of Hox genes are expressed at different levels of the embryonic trunk, thus conferring positional identity along the AP embryonic axis. The Hox genes activate the secretion of  FGF and TGF-βsuperfamilies.

Specific combinations of  Hox genes are involved in positioning the vertebrate limb fields. Hoxc6, Hoxc8, and Hoxb5 is expressed in the LPM, determines the forelimb (or pectoral fin) development. Mice lacking the Hoxb5 gene or if mice are mutated for Hoxb5, the shoulder girdle slightly shifted to the posterior region. This confirms a role for Hoxb5 in allocating the forelimb field.

Now let’s talk about the Regulation of Hox gene expression

Three genes regulate the Hox expression.

retinoic acid receptors (RARs),

the Krox20 gene,

members of the Pbx/Exd family of cofactors.

Retinoic acid (RA) is involved in controlling Hox gene expression in the lateral plate mesoderm at the time at which the limb fields are determined.  Apart from other hox, the hoxb8 gene is involved in initial anterior-posterior polarity of the limb bud. Inhibition of retinoic acid activity in the chick embryo downregulates the expression of the Hoxb8 gene.

GDF11 is a  TGF-β factor. GDF11 plays a role in the Anterior-posterior (AP) patterning of the axial skeleton. Gdf11-deficient mice show abnormality in the location of the forelimb and hind limb. The defect can be corrected with increased expression of the hox gene, this is suggested that GDF22 works upstream to the hox gene. These defects are correlated with alterations in patterns of Hox gene expression, which suggests that GDF11 acts upstream of the Hox genes specify positional identity along the AP axis.

11.4.      Anterior-posterior axis specification

Anterior-posterior axis specification is determined by sonic hedgehog. After the limb bud formation, sonic hedgehog protein expressing area is called a Zone of polarising activity (ZPA). Sonic hedgehog work as morphogen gradient. The zone of polasising activity of the limb bud has the pattern-organizing capability. The Sonic hedge hog gene is transcribed by Hox genes. Hoxb8 and dHAND gene product work as a transcription factor for Sonic hedge hog.  The level of Sonic hedgehog is sustained by reciprocal induction between the ZPA and the AER, in which Sonic hedge hog induces the AER to produce FGF4 and FGF8 to maintain its own expression.

The anterior digits 3, 4 and 5 are specified by a temporal gradient of Sonic hedgehog. Digit two is specified by a long-range diffusible form of Sonic hedgehog and Digit one does not require Sonic hedgehog. Interdigital tissue is present between the two-digit during limb development the digits get separated by apoptosis in interdigital tissue. The BMP signals responsible for the interdigit apoptosis as they are expressed in the interdigit tissue and blocking BMP signalling will prevent interdigital apoptosis, leads to syndactyly (fused digits). When the BMP signalling is blocked in the interdigital tissue it prevents apoptosis interdigital tissue. This leads to syndactyly (fused digit) condition.

Shh (Sonic hedgehog) signalling

Sonic hedgehog activates Gli. Gli is a Zinc-finger transcription factor. Without Sonic hedgehog, Gli2 and Gli3 act as a repressor and travels to the nucleus to repress the Sonic hedgehog effect. Sonic hedgehog cleaves the Ci/Gli3 repressor complex and thus the Gli is not cleaved and it now works as the activator. Gli activates the expression of the HoxD gene along the A/P axis. The inactivation of Gli3 repressor leads to the condition of polydactyly, in which many digits are formed abnormally.

11.5.      Dorsoventral Axis specification

The Dorsoventral signalling centre present in the dorsal ectoderm that is significant for patterning of ventral flexors muscles and dorsal extensors muscles. The critical morphogen considered for Dorsoventral polarity is the Wnt7a, expressed in ectodermal cells to induce the expression of Lmx1 gene in the underlying mesoderm that specifies the dorsal phenotype. The Wnt7a is both critical and sufficient to dorsalize the limb and its deficiency or inactivation may lead to a genetic defect named as Nail-patella syndrome.

The cellular origin during limb development in the form of Limb cartilage and bone cells are derived from LPM. It leads with the help of BMP morphogens BMP2 and BMP4. BMP2 and BMP4 are crucial to achieving chondrogenesis and later bone formation. Skeletal muscles of the limb are derived from somites (myotome) and dermal cells from the dermomyotome.