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.      The proximal-distal axis of the 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 polarizing activity of the limb bud has the pattern-organizing capability. The Sonic hedgehog gene is transcribed by Hox genes. Hoxb8 and dHAND gene product work as a transcription factor for Sonic hedgehog.  The level of Sonic hedgehog is sustained by reciprocal induction between the ZPA and the AER, in which Sonic hedgehog 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.

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