Mondia JP et al. (2011), Long-distance signals are required for morphoge...

XB-ART-44193

PLoS One 2011 Jan 01;69:e24953. doi: 10.1371/journal.pone.0024953.

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Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation.

Mondia JP , Levin M , Omenetto FG , Orendorff RD , Branch MR , Adams DS .

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BACKGROUND: With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration. METHODS AND RESULTS: Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis. CONCLUSIONS: We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.

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Species referenced: Xenopus laevis
Genes referenced: hpse not rpsa runx2

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	Figure 1. Illustration of the tadpole tail and techniques employed.(A) A stage 40 Xenopus tadpole shown 2â€“3 hours after amputation of one-third to one-half of the tail. (B) Enlargement of tail in A showing the regions targeted by the laser. Along the DV axis are the dorsal somite (dorSom); shoulder spinal cord (shSC), notochord (Noto), and ventral somite (ventSom). Along the AP axis of the spinal cord are anterior spinal cord (antSC); posterior spinal cord (postSC); and shoulder spinal cord (shSC). Scale barâ€Š=â€Š250 Âµm (C) Schematic of laser targeting setup. Pulses from an fs Ti:sapphire laser pass a shutter and neutral density filter (ND) before entering an inverted microscope housing a dichroic mirror (DM), microscope objective, and short pass filter (SPF). The tadpole sits atop a motorized x-y stage and is illuminated by white light (WL). The computer controls the shutter open/close duration and stage, while synchronously monitoring the specimen with a CCD camera. (D) Example of the five images that were recorded for each tail using 4Ã— and 10Ã— microscope objectives. Top to bottom they are: low magnification image before laser treatment, scale barâ€Š=â€Š150 Âµm; high magnification image used to focus and aim, scale barâ€Š=â€Š50 Âµm; high magnification record of the location and number of laser insults; high magnification image of laser damage; low magnification image of damage. (E) Image of a regenerated control tail including the position of the amputation plane (blue line) and the positions of the nine landmarks used for the Geometric Morphometrics analysis. Scale barâ€Š=â€Š1 mm.
	Figure 2. Histology of laser-pulse induced damage.(A) Sequential 8 Âµm sections through a region damaged by a single pulse. The extent of damage was two sections. Red stars indicate the notochord, which was not visibly damaged by the laser pulse. In contrast, the spinal cord was dramatically damaged; green arrows point at the undamaged spinal cord; red arrows point to damage. Scale barâ=â500 Âµm. (B) Section showing damage in ventral somite after 15 insults. (C) Section showing damage in the shoulder spinal cord after 15 insults. (D) Composite of a tail with particularly small melanocytes. (E,F) Sequential sections from the tail shown in D illustrating that the extent of damage/ablation depends strongly on the size of the melanocyte. In this example, damage is almost entirely restricted to the melanocyte itself (green arrow: undamaged melanocyte; red arrow: ablated melanocyte). (G) Image of a wound 8 days after healing at 22Â°C. The yellow arrowhead points to the cluster of dark pigment spots that appear at the site of laser damage. The red arrows point to the edges of what appears to be a scar formed where the pulses were delivered. Scale barâ=â100 Âµm. (H) Epifluorescence (Î»exâ=â488) image of the wound in G. The arrow points to the autofluorescence emitted by some component of the pigmented cluster. (I) The relationship between the extent of a lesion (gray diamonds) and the damage per laser insult (black circles) as a function of number of insults. The extent of a lesion is calculated by number of section showing damageÃ8 Âµm per section.
	Figure 3. Phenotypes of tails damaged at the regeneration bud and at different levels along the DV axis of the shoulder region.Dorsal is up and posterior is to the right in all images. Typical phenotypes of regenerated tails imaged at stage 48. In all panels, the blue line indicates the amputation plane and the orange arrowhead indicates the position of laser-induced damage. Scale barâ=â1 mm. (A) Control tail showing the normal shape of the regenerate. (B,C) Insults to spinal cord show normal development of the tail after 4 hours post laser (hpl) and 9 days post laser (dpl). (D) Image of regenerate after ablation of cells in the regeneration bud (RB: see inset). No observable difference was found when compared to the controls. (E) Ablation of dorsal somite (dorSom) cells does not affect regenerate shape. (F) Ablation of cells in the shoulder spinal cord (shSC) leads to an upward bend. (G, H) Targeting cells of the notochord (Noto) or ventral somites (ventSom) has no effect on regenerate shape. Dark lines in F and H are the staples used to hold tadpoles flat during imaging.
	Figure 4. Phenotypes of tails damaged at different sites along the AP axis of the spinal cord.Dorsal is up and posterior is to the right in all images. All tails were damaged at stage 40 and are shown at stage 48. Orange arrowheads point to the location of laser damage; the blue line indicates the amputation plane. Scale barâ=â1 mm. (A) The typical upward bend phenotype of tails damaged in the shoulder spinal cord (shSC). (B) Regenerate for tail damaged in the posterior spinal cord (postSC). (C) Image of the more severe phenotype caused by ablation of the anterior spinal cord (antSC). (D) Dorsal view of the tail shown in C illustrating the lateral bending of tails damaged in the antSC. (E and F) A tail damaged at two sites, the antSC and the shSC. This tail shows both characteristics of tails damaged at either shSC or antSC, including a simple upward bend, and LR bending. (G) The âpigtailâ spiraling at this tail tip is unique to tails that have been damaged at both antSC and shSC.
	Figure 5. Procrustes profiles of tail morphology after laser treatment.The nine points in all profiles represent the average position of the landmarks used to describe the shape of the regenerate (see Fig. 1E). In all cases the control refers to tails that have regenerated after amputation and all other profiles refer to regenerates after amputation and laser treatment. The numbers next to the legend are the probability that the profile will have that shape. If the P value is <0.05 then we assume the shape is statistically different from the control. The profiles and P values were calculated using Morphometric Geometrics as described in Supporting Information S1. (A) Compares the Procrustes profiles for insults to the shoulder spinal cord (shSC) for different hours post amputation (hpa). (B) Insults to the regeneration bud (RB) are shown not to affect regeneration. (C) For insults along the DV axis, only insults to the shSC and notochord (Noto) were significantly different from the control. Insults to the dorsal somite (dorSom) and ventral somite (ventSom) produced similar regenerates as the controls. (D) Profiles of insults along the AP spinal cord axis. The further anterior the damage, the greater the difference from the control. Even larger shape changes were observed in regenerates that had laser damage at two locations, anterior spinal cord (antSC) and shSC. * indicates p<0.01, indicates p<0.05, * indicates p<0.1.
	Figure 6. Model of morphogenetic information flow in regenerating tails.(A) An intact stage 40 tadpole showing the complete information distribution along the tail. (Bâ€“F) A chart of morphogenetic information flow, where the first column shows schematic representations of the information flow, the second column shows the Procrustes fits for tails damaged at specific sites compared with the control, and the final column gives the Procrustes distance between the two shapes. (B) A tadpole with an amputated tail. This diagram shows the flow of information (green arrow) that has been activated by the amputation. The origin of this information is the undamaged tail immediately anterior to the amputation plane. This is equivalent to the control situation. (C) The flow of morphogenetic information in a tail damaged at one site, close to the amputation plane (upper and lower green arrows). These two sources of information are from similar levels along the AP axis, thus carry morphogenetic information that is essentially the same, leading to only a slight affect on the shape of the regenerate. This is equivalent to damaging the shoulder spinal cord (shSC). (D) The information from a more anterior damage site differs more from the information at the cut plane, thus introducing â€œconflictingâ€ information to the regeneration process, and causing significant variation in shape of the regenerate, like that caused by damage to posterior spinal cord (postSC). (E) Damage far from the amputation plane will lead to the presence of morphogenetic information even more in conflict with that at the amputation plane, thus causing a severe affect on shape, like damage to anterior spinal cord (antSC). (F) The flow of morphogenetic information in a tail damaged at two sites. This panel illustrates how information from far anterior conflicts with both the amputation plane and the information from the other damage site, causing different and more severe changes in shape than either site alone, i.e., the greatest Procrustes distance.

References [+] :

Adams, The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis. 1990, Pubmed, Xenbase