Reprogramming of Positional Information in Blastema Cells from the Axolotl Regenerating Limb

Introduction

One of the primary goals in modern-day medicine is to determine how to regenerate tissues that have been lost or damaged due to aging, injury, or disease so that the human lifespan can be extended. By definition, regeneration refers to the regrowth of a damaged or missing organ from its remaining tissue. The field of regenerative medicine has taken three main approaches in order to research the effectiveness of different techniques in promoting regeneration. These methods include the implantation of stem cells to form new structures, the implantation of cells with a scaffold that can guide the direction of development, and the induction of cells in the body to replace missing or damaged structures. Although numerous advances have been made, the field of regenerative medicine still lacks the knowledge needed for regenerative techniques to be successful in practice (Brockes and Kumar 2005). To learn more about the steps being taken in today’s research, click here to visit the website dedicated to the NIH Center for Regenerative Medicine (NIH 2013).

Due to its ability to regenerate complex structures, the axolotl, Ambystoma mexicanum, is an ideal model for pinpointing the factors that regulate regeneration, more specifically limb regeneration. The axolotl is a type of urodele that originates from a lake beneath Mexico City (National Geographic 2014). It is best known for its ability to retain juvenile traits in adulthood, an occurrence referred to as neoteny (Developmental), and its ability to regenerate complex structures, such as the limbs and spinal cord. To observe the axolotl in its natural habitat, click here and take a look at the video Axolotl: Documentary on the Bizarre Mexican Salamander (Elmer 2013).

Image of Ambystoma mexicanum obtained from National Geographic.

Image 1. Image of Ambystoma mexicanum obtained from National Geographic.

Why choose the axolotl as the ideal model organism?

Unlike other vertebrates, the regenerative capability of salamanders is not lost at the end of the embryonic stage. This ability is carried into adulthood, allowing salamanders to regenerate complex structures that have been lost due to injury or some other event. It has been shown that salamanders’ regenerative capacity is due to the presence of tissue-specific stem cells that can generate cells belonging to that specific tissue. Although planarians are also capable of regenerating complex body structures using pluripotent stem cells that can form any tissue in the body, the tissue-specific stem cells from the salamander more closely resemble stem cells found in humans (Tanaka 2011). Since many biological pathways and signaling processes are conserved in tetrapods, it has been suggested that humans have the potential to regenerate complex structures using the same mechanism as salamanders. While humans can only form scar tissue and regenerate fingertips as of now, many researchers believe that the axolotl can be utilized to discover the processes leading to regeneration, which would hopefully lead to the induction of regeneration in human tissues (McCusker and Gardiner 2011).

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Image 2. Unlike planarians, which possess pluripotent stem cells that can differentiate into any tissue in the body, axolotls possess tissue-specific stem cells that can only give rise to that specific type of tissue. It has been proposed that human stem cells are more closely related to the tissue-specific stem cells found in salamanders, explaining why the axolotl is often chosen as the ideal model in regeneration studies. Image obtained from EuroStemCell.

What discoveries have been made in axolotl limb regeneration thus far?

Several models that explain the factors affecting limb regeneration in the axolotl have been proposed, one of which includes the Accessory Limb Model. This model suggests that the regeneration process is initiated by signals that result from a recent injury. After the wounded surface is covered with epithelial tissue, nerve signals cause dermal fibroblasts around the wound to dedifferentiate, migrate to the nerve, and form a blastema. Since a blastema is a group of mesenchymal stem cells that possess regeneration potential, the division of these cells eventually leads to the replacement of the missing limb (McCusker and Gardiner 2011). In other words, limb regeneration in the axolotl depends on three signaling pathways: (1) wound signal that leads to wound healing without scar formation; (2) nerve signal that leads to blastema formation; (3) blastema interactions that establish positional patterning and lead to the formation of a new limb (Gardiner 2011).

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Image 3. Image of a blastema that leads to limb regeneration in the axolotl. Signals between the nerve and wound epithelium cause fibroblast cells to dedifferentiate and migrate to the nerve so that a blastema can form (Brockes and Kumar 2005)

Purpose of Study

The purpose of the following study was to investigate how the limb pattern is reestablished from blastema cells. More specifically, the researchers grafted blastema cells in different regions and stages of blastema development from axolotls expressing Green Fluorescent Protein (GFP) to non-GFP hosts in order to determine the fates of these blastema cells during limb regeneration (McCusker and Gardiner 2013). The video below gives a brief overview of how grafting experiments that lead to induction of ectopic limbs are conducted with the axolotl (Science). The researchers hope that a better understanding of the mechanisms involved in regeneration using blastema cells will lead to advancements in the future that will allow the same regeneration process to occur in humans.

Description of Grafting Methods

As mentioned previously, the study seeks to address the question of how limb patterning is reestablished after grafting blastema cells in different regions (apical vs. basal) and stages (early bud vs. late bud) of development from GFP axolotl donors to non-GFP axolotl hosts (McCusker and Gardiner 2013). When using early bud (EB) blastemas, donor tissue was obtained from either proximal (near the shoulder) or distal (near the wrist) amputation sites in GFP donors and then grafted to proximal or distal amputation sites in non-GFP hosts. Although the same procedure was followed for late bud (LB) blastemas, the grafting experiments used either the apical or basal regions of the LB blastema. While the apical regions were obtained from the tips of LB blastemas lacking blood vessels, the basal regions were obtained from the region of LB blastemas closest to the stump tissue. The differences between early and late bud blastemas can be observed in the figure below (Figure 1).

Figure 1

Figure 1. A. Graft of early bud (EB) blastema. The thick blue dotted line represents the boundary between stump tissue and EB blastema. B. Graft of late bud (LB) blastema. The apical region contains very few blood vessels while the basal region shows much more evidence of vasculature (McCusker and Gardiner 2013).

Study Findings

Can early bud blastema cells acquire new positional identity when grafted to a new amputation site?

In the first part of the study, early bud blastemas from proximal donor amputation sites were grafted to distal host amputation sites in order to determine if early bud blastema cells have stable positional information or are labile in regards to positional identity. While proximal EB blastemas with stump tissue resulted in proximal-distal patterning duplications when grafted to distal host amputation sites (Figure 2A), grafts of proximal EB blastemas lacking stump tissue resulted in normal proximal-distal patterning along the limb (Figure 2B and 2C).  The inclusion of stump tissue in EB blastema cells resulted in patterning duplications by providing proximal-distal information corresponding to the donor site from which the blastema cells were grafted. These results suggest that early bud blastema cells are labile and acquire positional identity by interacting with the cells present at the distal host amputation site.

On the other hand, when EB blastema cells were grafted from a distal donor amputation site to a proximal host amputation site, normal proximal-distal patterning was observed, regardless of whether or not stump tissue was present (Figure 2D and 2E). Due to conclusions in previous experiments, this result was expected.

figure 2

Figure 2. A. Results obtained when proximal EB blastema with stump is grafted to a distal amputation site. Red arrows mark distal amputation plane on host limb. Green arrows indicate the duplicated elbow joint and radius/ulna. B-C. Results obtained when proximal EB blastema without stump is grafted to a distal amputation site. No duplicated proximal-distal patterns were observed. Grafted cells (colored green) contributed to connective tissue (1), muscle (2), and cartilage (3). D. Results obtained when distal EB blastema with stump is grafted to a proximal amputation site. Normal proximal-distal patterns were observed in the regenerated host limb. E. Results obtained when distal EB blastema without stump is grafted to a proximal amputation site. Normal proximal-distal patterns were observed in the regenerated host limb. The grafted cells contributed to the formation of nerves (1 in E), bone (F), fibroblast-like cells in dermis (G), cartilage and its surrounding connective tissue (H), and muscle (I) (McCusker and Gardiner 2013).

What other evidence supports the finding that early bud blastema cells are labile?

To further assess the lability of EB blastema cells when grafted to new host amputation sites, the expression of positional marker genes was measured in regenerating limbs by grafting distal forelimb EB blastemas containing GFP to either forelimb or hindlimb amputation sites in non-GFP hosts. After allowing the blastemas to develop, the GFP grafted cells were separated from the non-GFP host cells via fluorescence-activated cell sorting (FACS) so that the expression of forelimb and hindlimb marker genes could be measured (Figure 3A).

As it has already been noted in class, forelimb buds yield higher Tbx5 expression while hindlimb buds yield higher Tbx4 expression. Due to this difference in Tbx5/Tbx4 expression, the activities of these positional markers were measured in order to assess if forelimb blastema cells can be induced to express Tbx4 upon grafting to a hindlimb stump in a host amputation site. While the grafting of GFP+ forelimb blastema cells to GFP- forelimb stumps resulted in low levels of Tbx4 expression, the grafting of GFP+ forelimb blastema cells to GFP- hindlimb stumps resulted in a significant increase in Tbx4 expression and elimination of Tbx5 expression in the donor cells (Figure 3C and 3D). This increase in TBX4 expression and elimination of Tbx5 expression indicate that EB blastema cells can change their pattern of gene expression when grafted to a new amputation site.

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Figure 3. A. Process of separating GFP+ (graft) cells from GFP- (host) cells via fluorescence-activated cell sorting (FACS). B. Histogram showing the fold change of expression in FL blastema cells relative to HL blastema cells. Tbx4 and Tbx5 were validated as markers using q-rtPCR. C. Histogram showing relative expression of Tbx4. FL+ and HL+ represent GFP+ FL grafts to FL and HL, respectively. FL- and HL- represent GFP- FL host tissue and GFP- HL host tissue, respectively. D. Results from RT-PCR performed on graft and host cells for Tbx4, Tbx5, and GAPDH. Note: Tbx4 tends to show higher expression in the hindlimb while Tbx5 tends to show higher expression in the forelimb (McCusker and Gardiner 2013).

How similar are early and late bud blastema cells in regards to their ability to acquire new positional identity when grafted to a new amputation site?

After demonstrating how early bud blastema cells change their positional identity and gene expression along the proximal-distal axis by interacting with cells adjacent to the host amputation site, the researchers wanted to determine if there are differences in the lability of the apical and basal regions of the late bud blastema. When the basal region of a proximal LB blastema was grafted to a distal amputation site, a limb with duplicated proximal-distal patterning resulted  (Figure 4A). However, when the apical region of a proximal LB blastema was grafted to a distal amputation site, a limb with normal proximal-distal patterning resulted (Figure 4B). Considering the results obtained from the previous experiments (Figure 2), the grafting experiments using the basal region of the LB blastema cells produced the same results as those using the EB blastema cells containing stump tissue while the grafting experiments using the apical region of the LB blastema cells produced the same results as those using the EB blastema cells lacking stump tissue. These observations suggest that the apical region of the LB blastema remains undifferentiated and can acquire a new proximal-distal identity by interacting with cells adjacent to the host amputation site and that the basal region of the LB blastema maintains its donor proximal-distal identity upon being grafted to the new amputation site.

figure4

Figure 4. A. Results obtained when a proximal LB blastema from the basal region is grafted to a distal amputation site. Green arrows mark the duplicated elbow joint and radius/ulna. B. Results obtained when a proximal LB blastema from the apical region is grafted to a distal amputation site. No proximal-distal pattern duplications were observed. C. Results obtained when a distal LB blastema from the apical region is grafted to a proximal amputation site. No proximal-distal pattern duplications were observed. D. Results obtained when a distal LB blastema from the basal region is grafted to a proximal amputation site. No proximal-distal pattern duplications were observed. Grafted cells from both the apical and basal regions of the LB blastema contributed to the formation of blood vessels (E), cartilage (F), connective tissue (G), nerve-associated cells (H), muscle (I), and dermis cells (J) (McCusker and Gardiner 2013).

How is the lability of early bud blastema and apical late bud blastema cells maintained?

After assessing the lability of both early and late bud blastema cells, the researchers sought for the mechanism by which blastemas maintain their undifferentiated state until regeneration of the entire limb is complete. To determine this process, the researchers performed additional grafting experiments where either blastema or differentiated cells were grafted to a wound site with access to a nerve. When differentiated posterior skin cells and basal LB blastema cells were grafted to an innervated wound site, the formation of ectopic limbs was observed (Figure 5A and 5F, respectively). However, both grafted EB blastema and apical LB blastema cells failed to produce ectopic limb structures when grafted to the innervated wound site (Figure 5B and 5D, respectively).  When these EB blastema and apical LB blastema cells were denervated in the donor limb three days prior to grafting, they were able to form ectopic limb structures upon grafting to the innervated wound site (Figure 5C and 5E, respectively). These results suggest that signals from a nerve near the wound site are required to maintain blastema cells in an undifferentiated state.

figure5

Figure 5. A. Results obtained after posterior skin was grafted to an innervated wound on the anterior side of the arm. Ectopic limb was observed. B. The grafting of EB blastemas to an innervated wound did not result in the formation of limb structures. C. The grafting of EB blastemas from a denervated donor limb to an innervated wound resulted in the formation of an ectopic limb. D. The grafting of apical LB blastemas to an innervated wound did not result in the formation of limb structures. E. The grafting of EB blastemas from a denervated donor limb to an innervated wound resulted in the formation of an ectopic limb. F. Results obtained after basal LB blastemas were grafted to an innervated wound on the anterior side of the arm. Ectopic limb was observed (McCusker and Gardiner 2013).

Concluding Remarks

In order for regeneration to occur, cells in the amputated limb must replace the more distal limb structures that have been lost. Thus, the cells at the tip of the amputation site acquire distal positional information by interacting with the more proximal cells in the wound site (Figure 6A). This study shows how EB blastema cells and the apical region of LB blastema cells are labile and can reprogram their positional identities. The finding that the basal region of LB blastema cells does not have the same ability to reprogram its positional identity suggests that its cells are already differentiated and are maintained in a stabilized state (Figure 6A). Although the exact mechanism by which positional information is regulated during regeneration remains unknown, it can be concluded that the stability of positional identity in blastema cells is most likely maintained by signals between the wound site and nerve (Figure 6B).

figure6

Figure 6. A. (Top image) While the early blastema cells maintain a distal (“D”) identity, an intermediate (“I”) identity is acquired upon interaction with proximal (“P”) cells; (Bottom image) The distal cells in the early and apical late bud blastemas remain labile (“L”) while the proximal cells near the stump and basal regions become stable (“S”); B. The loss of nerve signals causes premature stabilization of differentiated cells (McCusker and Gardiner, 2013).

Critiques of Experimental Study

From an undergraduate student’s perspective, the experimental study appears very thorough and detailed. The methods section was logically organized and used simple language that allowed the readers to easily understand what was being done in each part of the study. The researchers also took the time to describe any discrepancies in the results. For example, if the current results differed from those in previous experiments, the researchers assessed the discrepancies and suggested any changes in the procedure that could have led to different results. Although it was a very strong paper with well-supported evidence, the results would be more valid if additional grafting experiments were conducted. In this study, only 5-8 grafts were conducted for each individual experiment. The researchers also failed to mention the mechanism by which they believe blastema cells reprogram their positional identity and did not offer any suggestions for future studies.

References

Brockes, J.P, and Kumar, A. 2005. Appendage regeneration in adult vertebrates and implications for regenerative medicine. Science. 310(5756): 1919-1923.

Developmental Biology Interactive. Posting date unknown. A webpage that discusses the mechanisms underlying neoteny in axolotls. Available from http://www.devbio.biology.gatech.edu/?page_id=497 [accessed 3 April 2014].

Elmer, Roddy. 3 December 2013. Axolotol: Documentary on the bizarre Mexican salamander [Video file]. Retrieved from https://www.youtube.com/watch?v=4wcdy9FehaI [accessed 3 April 2014].

Gardiner, D.M. 2011. A resource that explains the implications of the Accessory Limb Model. Available from http://regeneration.bio.uci.edu/accessory-limb-model/ [accessed 3 April 2014].

McCusker, C.D., and Gardiner, D.M. 2011. The axolotl model for regeneration and aging research: a mini-review. Gerontology. 57(6): 565-571.

McCusker, C.D., and Gardiner, D.M. 2013. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum). PLos ONE 8(9): e77064.

National Geographic. 2014. A resource that describes the background and characteristics of the axolotl. Available from http://animals.nationalgeographic.com/animals/amphibians/axolotl/?rptregcta=reg_free_np&rptregcampaign=20131016_rw_membership_r1p_us_dr_w# [accessed 3 April 2014].

NIH Center for Regenerative Medicine. 2013. A resource that describes the current-day research in the field of regenerative medicine. Available from http://crm.nih.gov/ [accessed 3 April 2014].

Science Channel. 22 November 2010. Superhero science- Limb regeneration [Video file]. Retrieved from https://www.youtube.com/watch?v=EsCSwVx3GvA [accessed 3 April 2014].

Tanaka, Elly. 2011. A resource that explains the meaning of regeneration and how it works in salamanders [online]. Available from http://www.eurostemcell.org/factsheet/regeneration-what-does-it-mean-and-how-does-it-work [accessed 3 April 2014].

 

 

 

 

 

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