Hedgehog Signaling Controls Dorsal Ventral Patterning and Induction of Axolotl Tail Regeneration

This blog is based off the paper “Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration” by Ester Schnapp, Martin Kragl, Lee Rubin, and Elly M. Tanaka in the Development Journal, 2005.


Have you ever seen a newt or salamander that had it’s limb or tail severed? Did you notice that hours later that the limb was growing back?
The Caudata, an order of tailed amphibians, are especially notorious for rapid regeneration of  limbs, tails, jaws, eyes and a variety of internal structures. One such organism is the axolotl, also called the Mexican salamander.

Background on the Axolotl

The axolotl is a neotenic salamander, meaning that these amphibians have retention of juvenile characteristics in to the adult life stages. The larvae fail to mature and thus remain gilled and aquatic. These salamanders are specifically found in lakes found underneath Mexico City hence the name “The Mexican Salamander”. Lately (2010), the axolotl has become near extinct due to urbanization of Mexico City. The salamanders live in an aquatic environment that stays at 68°F and can drop down about 7 degrees in the water. These organisms sexually mature and become adults around 18-24 months and usually possess external gills and a caudal fin that runs the length of their body.  Axolotl’s can live up to 15 years on a diet consisting of worms, insects, and small fish.

The main characteristic of these salamanders that causes it to be a model organism is it’s amazing healing abilities. The axolotl can regenerate entire lost appendages in a period of months and in certain cases, parts of the brain (Lee et. al. 2012). Also, this salamander can readily receive transplants from other organisms and able to fully function as if they were their own.

A study published by Schnapp et al. studies the effects of coordinated growth and patterning of regenerating tissues in the axolotl. The role of not only Sonic Hedgehog is studied but also Pax6, Msx2, and many other genes and transcription factors.

Introduction

When an axolotl has their tail severed, the process of regeneration involves regrowth and patterning of multiple tissue types including part of the spinal cord, muscle, cartilage, skin, and fins. After amputation and some wound healing, the spinal cord grows out as a tube that is surrounded by a myriad of progenitor cells. Implanting experiments by Holtzer suggests that cartilage is induced by the ventral half of the spinal cord and that the mature spinal cord with surrounding tissue is regenerated upside down (Schapp et al. 2005). This work also implies that dorsal-ventral patterning information is seen in the spinal cord and allows tissue to regenerate. Dorsal-ventral patterning is required for proper growth and development.

The neural tube is surrounded by different regions that are defined by a series of homeodomain and paired-boxes that contain transcription factors. The dorsal most domain is defined by Msx1 and 2, the dorsalateral cell by Pax7, and lateral domains by Pax6. Sonic hedgehog (Shh) is a factor that is expressed in the notochord and floor plate and induces ventral neural tube cell types in a concentrated manner. Bone morphogenetic proteins (BMPs) are expressed in the ectoderm and dorsal roof plate. These proteins are antagonists of Shh, but BMP4 and BMP7 activate expression in Msx1, Pax7, and Pax6 in the dorsal and lateral neural tube (Lee et al 2012). In low concentrations of Shh, Msx1 and Pax7 are blocked but elevate Pax6 expression in the floor plate of neural tube. However, when concentrations of Shh are high, Pax6 is inhibited.

Even though these morphogens are mainly discussed during development, they are not just restricted to the neural tube. They also play a role in cell proliferation, patterning, and cell-type specification. Shh induces the expression of sclerotome markers such as Pax1 and Sox9 which are essential for cartilage formation. Also, Shh regulates myogenic precursors such as Myf5 for muscle development and induces proliferation of somitic mesoderm. Shh negatively signals its own signaling by upregulation of the Shh receptor Patched1. All of these interactions are a basis of studying the regeneration of limb buds.

Goal: To investigate the DV patterning in the axolotl spinal cord and how the spinal cord can communicate patterning information to the regenerating spinal cord and surrounding tissue. In this experiment, they are demonstrating whether Shh, Pax6, Pax7, and Msx1 are expressed in their domains within the mature Axolotl spinal cord as well as the ependymal tube. Patched 1 expression further indicates that hedgehog signaling occurs within the spinal cord and in blastocyst cells. Blocking Shh signaling through the drug cyclopamine, it is shown that Shh is not only required for DV patterning but also overall tail regeneration. Proliferation of blastocyst cells and Sox9 expression depends on hedgehog signaling.

Questions: What is the function of Shh in the establishment of DV identity of the regenerating tail? Is interfering with the DV pattern in the regenerating spinal cord effect DV organization of the tail? Is Shh necessary for ependymal cell proliferation and can Shh act as a mitogen?

Evidence for Shh Signaling in Limb Regeneration

  1. How marker genes and transcription factors seen in DV patterning

To determine the DV patterning of the axolotl spinal cord, the marker genes were analyzed in the developing neural tube.

Fig1. The spinal cord was removed over the length over several segments and was allowed to heal over several days. Shh is clearly expressed in the floor plate of the differentiated spinal cord (A) and in the ventral most ependymal cells. Shh expressed only in the spinal cord clearly seen by the blue dye (B, C). Pax6 is expressed in the lateral cells of the axolotl spinal cord and of the ependymal cells (D, E). Pax7 is located in the dorsolateral domain of the ependymal tube shown by the bright pink highlighting (F, G). Msx1 is clearly expressed in the roof plate of the differentiated spinal cord (H-J).

Shh, Pax6, Pax7, and Msx1 are described as distinct markers of dorsoventral neural progenitor cells. All these markers were expressed in the mature axolotl spinal cord and in the ependymal tube in DV domains.

Gene and protein analysis on tissue sections indicated that the progenitor cells in the mature spinal cord show patterning of DV neural tube markers. The same DV pattern is present in the ependymal tube throughout axolotl tail regeneration.


2. Is Shh required for tail regeneration?

In order to test this, the chemical inhibitor cyclopamine was administered in the water and is used to block hedgehog signaling by antagonizing the Shh receptor Smoothened. In the presence of cylcopamine, axolotl tail regeneration was inhibited(Fig. 2A-G) but wound healing and fins formed properly (Fig 2. D-F).

Fig2. This examines whether cartilage progenitors in the early blastema express Sox9, and whether Sox9 is controlled by hedgehog signaling. Cylcopamine treatment is used and is seen to block axolotl tail regeneration but a hedgehog agonist clearly rescues this phenotype (A-C). The fin and ependymal tube do not grow back in D-F but a blastema is seen to be slightly forming. G represents the quantification of the lengths of the control experiment with cyclopamine-treated tail regenerates over time. The graphs show the regenerating tail lengths over time provided with error bars and standard deviations and dashed lines in A-F and H-J mark the amputation plane. The tails in I and J are indistinguishable.

Time was an important factor in which in the cycopamine-treated axolotls were able to regenerate 4 days post-amputation with a control in Figure 2D. Then, at later stages of regeneration, up to 14 dpa there was no cartilage or muscle differentiation. After 8 days of being treated with cyclopamine, the outgrowth of the ependymal tube stopped (Figure 2G)

3. Hedgehog signaling is necessary for the correct establishment of DV domains in the ependymal tube

The cyclopamine-treated regenerates for DV patterning show expansion of the dorsal spinal cord markers, Pax7 and Msx1 moving in to ventral regions (3A,D). With cyclopamine plus the agonist-treated regenerates, the Msx1 and Pax7 domains were restored and had a rescue effect (Fig 3B, E). Treatment of regenerating tails with the agonist alone did not produce morphological effects, but low concentrations of agonist eliminated Pax7 expression from the dorsal spinal cord (Fig. 3C) and Pax7-positive cells persisted in the presence of agonist.

Fig3. These figures show dorsal spinal cord domains in the presence of cyclopamine with rescue regenerates. Pax-7 antibody stainings were performed on the regenerate (A) which showed the dorsal ependymal tube is expanded ventrally and then on the rescued regenerate (B). C is the agonist-treated regenerate in which the ependymal tube is absent. Hoechst staining is the blue while Pax7 staining is in red in the in-situ hybridization of cyclopamine-treated regenerates (D, E).

4. Hedgehog Signaling is required for Sox9 expression in tail blastema

Fig5. Cyclopamine-treated blastemas with Sox9 and Pax7. A-C show Sox9 in situ hybridization, A being the control, B is cyclcopamine-treated, and C is agonist-treated regenerates. The columns in the graph represent the mean percent in Pax-7 positive blastema cells of the three treatments.

During development, Shh induces the expression of the early cartilage marker Sox9 in the sclerotome. It was found that Sox9 was expressed in a defined area of the blastema ventral to the spinal cord from 4dpa onward which is 2 days before cartilage differentiation. Sox9 expression was not detectable in cyclopamine-treated regenerates 6 dpa (Fig. 5A, B), while agonist-treated regenerates showed an increased expression of Sox9 and some dorsal blastema cells expressing the gene (Fig. 5C). It was concluded that no cartilage differentiation was observed in the dorsal blastema and the ventral cartilage appeared normal.

5. Hedgehog signaling controls blastema cell proliferation rather than ependymal cell proliferation

There was a reduction of size seen in the blastema in cyclopamine-treated regenerates and it was examined whether the reduction was due to apoptosis or a block in cell division. TUNEL staining of cyclopamine samples were indistinguishable suggesting that apoptosis was not the reason for the size reduction. To examine cell proliferation, BrdU labeling for 42 and 72 hours occurred starting 3 dpa. This led us to believe that there was a different effect of cyclopamine of proliferation of ependymal cells versus ventral blastema cells (Fig. 6A and 6B). Hodgehog is concluded to signal controls the proliferation of around 40% ventral tail blastema cells.

6. Shh has distinct activites on the limb versus tail blastema

Limb blastemas were treated with cyclopamine and the result was regenerates of normal length but lacking digits (Fig. 7) that is consistent with the expected defects of anteriorposterior (AP) digit patterning. Cyclopamine-treated limbs only affected the AP patterning of the digits.

Fig7. Cyclopamine treatment of regenerating axolotl limb only affects digit loss. (A) is the control and (B) is the regenerated cylopamine-treated limb, The arrow points to the regeneration of rod cartilage.

-Ectopic activation of hedgehog signaling in the absence of the spinal cord is not sufficient for tail regeneration

This was tested by removal of the spinal cord from the distal tip of the the tail and it was seen that subsequent tail amputation blocks growth until the spinal cord regenerates. The spinal cord

Fig. 8. Hedgehog signaling is not sufficient for tail regeneration without the spinal cord. (A) Mock operated axolotl tail shows normal 7-day regenerate. (B) Control tail with spinal cord removed does not regenerate. (C) Agonist-treated tail without spinal cord also does not regenerate. Arrows point to the distal tip of the spinal cord in all panels.

tip was removed from the axolotl tail and treated with Shh agonist (Fig. 8A-C). This shows that Shh alone will not rescue tail regeneration in the absence of the spinal cord. The other factors mentioned must be present for tail regeneration.

Strengths and Weaknesses

Strengths-

  1. This study proves it’s hypothesis in an efficient manner.
  2. Researchers perform a myriad of experiments to make sure cyclopamine is solely responsible for blocking limb regeneration and show the direct affects on the desired genes.
  3. The paper was easy to follow and provided sufficient information and figures as evidence.

Weaknesses-

  1. It focused too much on the affects of cyclopamine and did not show what could happen in the presence of other drugs.
  2. The paper was not extremely current but the other mentioned papers did help this flaw.

Why do we care?

This study could lead to possible research on limb regeneration methods in humans.

References

Lee, J., Gardiner, D. Regeneration of limb joints in the axolotl. US Library of Medicine. (2012).

Jhamb, D., Roa, N., Milner, D., Song, F., Cameron, J. Network based transcription factor analysis of regenerating axolotl limbs. BMC Bioinformatics. (2011).

Schnapp, E., Kragl, M., Rubin, L., and Tanaka, E. Sonic hedgehog signaling controls dorsalventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration. Developmental Journal. (2005)

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