Salamander Limb Regeneration Compared to Embryonic Limb DevelopmentNathan ZerwigIntroductionRegeneration of limbs is commonly seen in vertebrates such as salamanders, tadpoles, and fish species. This regenerative power is not seen in other vertebrates such as mammals and birds. What allows for salamanders to regenerate lost limbs and what prevents mammals from re-growing lost limbs? Answers may be found by comparing the processes of salamander limb regeneration and embryonic development.
Regeneration in salamanders requires adult tissues to form unspecialized regions of growth to reform the limb or part of the limb that was lost. Embryos require a specialized limb with specialized tissues be produced from unspecialized cells in a relatively simple system compared to the adult limb. Understanding the process of regeneration in vertebrate species and how this process compares to limb development in vertebrate embryos could lead to answers of why some vertebrates, such as humans cannot regenerate limbs. It appears that some of the processes and mechanisms involved in limb regeneration are similar to those of limb formation during embryonic development, but there are also some major differences between the two processes. This paper discusses some of the similarities and differences in processes and mechanisms seen in limb regeneration and development.The Blastema and Limb BudAfter amputation of the limb of a salamander, a small mass of cells called the blastema forms on the distal end of the amputated limb. This blastema contains mesenchymal cells that have a degree of pluripotency, allowing them to divide and reform tissues needed to regenerate the lost limb of the salamander.
The blastema can be considered analogous to the embryonic limb bud formed during embryonic development. Limb buds also use pluripotent cells, specifically progenitor cells, to form the tissues of the developing limb. Furthermore, similar transcription factors have been noted in the blastema and limb bud, suggesting again that these two structures may form limbs through similar pathways.Axis FormationRule of Distal TransformationRegeneration of an amputated limb of a salamander will produce a fully functional structure almost identical to the original without any extra parts or missing parts. This means that cells in the limb must have positional memory. The positional memory of the amputated limb is best demonstrated by the rule of distal transformation. This rule was demonstrated by researchers removing a salamander’s hand at the wrist, suturing the wrist of the limb into the trunk of the salamander, letting the sutures heal, and then cutting the sutured limb (Butler 1955).
The cut limb produced two new limbs each with hand elements. So, each cut portion of the limb grew elements that are distal relative to their position, but how exactly does the blastema produce the missing elements of the limb and how are axes maintained in regeneration?IntercalationResearch strongly suggests that cell-cell contact within the regenerating limb strongly influences proper regeneration of the missing limb. One theory on the mechanism of regeneration is that cells of the amputated limb and blastema regenerate a limb through intercalation. In intercalation of a regenerating limb, the blastema is thought to form the most distal elements of the limb and proximal elements are generated between the site of amputation and the blastema. Circumferential intercalation is also observed in salamander limb regeneration. A misalignment of the circumference of an amputated right limb and a blastema from a left limb resulted in the formation of three hands. The misaligned circumferences of the amputated stub and blastema resulted in circumferential intercalation between the misaligned anterior/posterior axes of the limb, forming two new limbs (Bryant et al.
1981). Intercalation is not observed in embryonic development of chick limbs. In the developing chick limb, cells are proposed to be added distally to the limb bud, with each new cell layer forming successively distal structures. Parts are not added between the most distal and proximal cells of the limb bud. Intercalation is a major mechanism in salamander limb regeneration but does not have a major role in embryonic limb development.Morphogen GradientsMorphogen gradient models have also been discussed as a mechanism for regeneration and development. In this theory, mesenchymal cells migrate to the amputated stump and induce morphogen release from the surrounding ectoderm cells. In development, the anterior limb bud cells express GL13, while 2(HAND2) is expressed in the posterior cells.
The dual expression of 2(HAND2) and HOXD13 along the proximal/distal axis of the limb produce a morphogen gradient of sonic hedgehog, maintaining the anterior/posterior axis (Galli et al. 2010). Sonic hedgehog also causes expression of gremlin, a BMP antagonist, that is thought to play a role in formation of distal structures of the limb. It is also thought that FGF could play a role in axis formation and maintenance in regeneration like as in limb development in embryos, but more studies need to be performed. Other transcription factors known from the limb development cascade have been observed in the blastema during regeneration. These include wnt5, dlx3, and sp9. More studies need to be performed to determine whether these factors have common roles in the processes of limb development and regeneration.Retinoic acid (RA) may also play role in maintaining the proximal/distal axis during regeneration.
RA was found to be a strong proximalizing agent when introduced to regenerating salamander limbs. RA can cause structures in the regenerating limb to break the rule of distal transformation and cause these structures to be identified as proximal. A cell surface molecule, prod1, was also identified to be present at higher concentrations in proximal elements of the regenerating salamander limbs than in the distal elements.
RA and the prod1 surface molecule are thought to play significant roles in maintaining the proximal/distal axis of the regenerating limb due to their effects and positionings on and in the regenerating limb.The MEIS family homeodomain transcription factors play a role in forming proximal limb structures in regeneration and limb development (Nacu and Tanaka 2011). FGF and BMP found in the distal limb portions inhibit MEIS factors. MEIS factors are also thought to induce prod1 in regeneration, forming a gradient along the proximal/distal axis. HOXA transcription factors also aid in the formation of the proximal/distal axis. In development, hoxa9-13 are expressed in order down the developing limb and each of these different HOXA transcription factors are required for formation of the upper arm, lower arm, and hand regions (Nelson et al.
1996). In limb regeneration, hoxa9-13 are all expressed in the early blastema, which suggests that distal portions of the forelimb are formed first, opposed to distal elements being formed last in development.Nervous Tissue in RegenerationIt was also found that nervous tissue of an amputated limb may play a role in regeneration. Nerve endings moved to under the epidermis of a normal limb can result in the formation of a blastema-like bump forming under the epidermis.
The accessory limb model successfully induced ectopic limb formation by grafting skin from the posterior of a salamander to an anterior wound innervated by an upper limb nerve (Endo et al. 2004). The accessory limb model experiments also found that hoxa13 is induced by nerves in the blastema. This hoxa13 is a major player in finger and hand formation, meaning that nervous tissue induces the most distal elements of a limb to form during regeneration. Newt anterior gradient factor (NAG) is also induced by nerve in the blastema. NAG as found to cause proliferation of blastema cells, but it is not yet determined whether it contributes to the determining of the proximal/distal axis of the regenerating limb.DiscussionIt appears that regeneration of amputated limbs of salamanders and limb development in embryos use similar mechanisms for growth and differentiation, but the processes also have their differences. It seems that both processes implement morphogen gradients to develop and maintain anterior/posterior and proximal/distal axes to some extent.
It is suggested that regeneration and development of limbs use GLI3, 2(HAND2), sonic hedgehog, and other factors to establish gradients along the limb used to form the axes. MEIS transcription factors associated with proximal/distal patterning during embryonic limb development were also discovered to play a role in patterning of regenerating limbs. HOXA transcription factors are also major proximal/distal axis formation factors in both processes, but the temporal expression of the different HOXA factors are different in the processes. There are also many differences between the mechanisms of regeneration and development, though.Regeneration of salamander limbs is thought to use the process of intercalation opposed to the continual addition of more distal structures during limb formation in embryonic development. Retinoic acid and prod1 are believed to participate in the maintenance of the proximal/distal axis in regeneration but are not thought to be used in development.
It was also found that nervous tissue of amputated limbs may contribute to the regeneration of that limb, supplying distal transcription factors to the blastema of the regenerating limb. ConclusionIt is apparent that there is still much unknown about the process of regeneration, and whether it uses similar processes of embryonic development. Further research into the mechanisms and machinery of regeneration would help to further understand just exactly how limbs are regenerated in vertebrates such as salamanders, and why other vertebrates cannot regenerate limbs. Once the process of regeneration is clearly understood, researchers may be able to implement the processes of regeneration in the medical field to regrow human structures.
REFERENCESBryant SV, French V, Bryant PJ. (1981). Distal regeneration and symmetry. Science 212:993–1002Butler EG. (1955). Regeneration of the urodele forelimb after reversal of its proximo-distal axis.
J. Morphol. 96:265–81Endo T, Bryant SV, Gardiner DM. (2004).
A stepwise model system for limb regeneration. Dev. Biol. 270: 135–45Galli A, Robay D, Osterwalder M, Bao X, B´enazet J-D, etal. (2010).
Distinct roles of Hand2 ininitiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet. 6:e1000901Nacu E, Tanaka EM. (2011). Limb Regeneration: A New Development? Annu. Rev. Cell Dev.
Biol. 27:409–40Nelson CE, Morgan BA, Burke AC, Laufer E, DiMambro E, et al. (1996). Analysis of Hox gene expression in the chick limb bud. Development 122:1449–66Discussion of Figure 6 in Nacu E, Tanaka EM. 2011.
Limb Regeneration: A New Development? Annu. Rev. Cell Dev. Biol. 27:409–40Figure 6 explains the polar coordinate model of circumferential intercalation as explained by Bryant et al.
and French et al. This model illustrates how the proximal/distal axis is maintained in regeneration while producing more distal cells of the limb. It also illustrates how circumferential intercalation and polar coordination can explain why supernumerary digits occur when a left blastema is placed on a right limb stump. Part A of figure six just shows the generalized arrangement of a limb from proximal regions to distal regions.
Circumferential numeric values are given in the diagram in a clockwise manner. The letters on the diagram represent the position of the cells along the proximal/distal axis. Part b diagrams the process of intercalation and distalization of cells in the regenerating limbs.
An amputated limb begins healing and cells of different circumferential values move to cause cell to cell contact of inconsecutive cells along the circumference of the limb. This is demonstrated by cells with the value of 1A, 4A, 7A, and 10A moving inward in the amputated limb. The asterisks in the diagram denote areas where intercalation between the touching cells of 1A, 4A, 7A, and 10A. These cells cause intercalation of cells 12B, 2B, 3B, 5B, 6B, 8B, 9B and 11B in a plane more distal to the A cells.
The missing cells of 1B, 4B, 7B, and 10B are then intercalated in the more distal B layer of cells, maintaining the circumferential positioning of the cells in each cell layers. This process is repeated down the proximal/distal axis of the limb forming continually more distal cell layers of the limb.Part c of the figure describes how a misalignment of the posterior/anterior axis of a limb stump of a right leg and the blastema of a left leg. The misaligned circumferential values cause intercalation of the missing values. On the left side of the regenerating limb, the 9 cell from the limb stump and the 3 cell from the left blastema come into contact. This signals an anterior posterior axis to the regenerating limb. Intercalation of all circumferential cells occurs to form an extra digit.
The same phenomenon happens on the right side of the regenerating limb, where the 9 cell of the left blastema comes into contact with the 3 cell of the limb stump. This intercalation of cells assigned to the missing circumferential values between the misaligned posterior/anterior axes causes the formation of two entirely new posterior/anterior axes on the regenerating limb. These two additional axes cause supernumerary limbs to form. One limb develops out of each of the anterior/posterior axes of the regenerating limb.This polar model displays the importance in cell-cell signaling in the regeneration of limbs.
The cells exchange information that allows the recognition of missing components along the circumference of the limb. This allows the cells to divide and intercalate the missing cells. The polar coordinate model also displays the rule of distal transformation, providing a mechanism for how the proximal cells of a regenerating limb form distal layers through intercalation.