December 1, 2006;
Characterization of Xenopus digits and regenerated limbs of the froglet.
Xenopus has 4 and 5 digits in a forelimb
, respectively. It is thought that their limbs and digits develop in Xenopus by mechanisms that are almost conserved from amphibians to higher vertebrates. This is supported by some molecular evidence. The 5''hoxd genes are convenient marker genes for characterizing digits in the chick and mouse. The anteriormost digit
is characterized by being hoxd13
-positive and hoxd12 (hoxd11
)-negative in the chick and mouse. In this study, we revealed that the anteriormost digit
of the Xenopus forelimb
-positive and hoxd11
-positive, that is, a more posterior
character than digit
I. The order of formation of digit
cartilages also suggested that Xenopus forelimb digit
identity is II to V, not I to IV. We have also been interested in the relationship between digit
identity and shh
. The anteriormost digit
develops in a shh
-independent way. A limb
treated with cyclopamine (a shh
inhibitor) has a gene expression pattern (hoxd11
-negative) similar to that in shh
-deficient mice, suggesting that a hindlimb
treated with cyclopamine has a digit
I character. However, a Xenopus froglet regenerate (spike
), which lacks shh
expression during its regeneration process, does not have such an expression pattern, being hoxd11
-positive. We investigated hoxd11
transcriptions in blastemas that formed in the anteriormost and posteriormost digits, and we found that the blastemas have different hoxd11
expression levels. These findings suggest that the froglet limb blastema
does not have a mere digit
I character in spite of shh
defectiveness and that the froglet limb blastema
recognizes its positional differences along the anterior
[+] show captions
Figure 2. Gene expression in the Xenopus developing hindlimb. A-E: Gene expression in the forelimb bud at the paddle stage (Nieuwkoop and Faber stage 52). A:Hoxa13 expression. Digit cartilage started to differentiate and hoxa13 expression was downregulated (compare with 3E). B:hoxd13 expression. In the anterior domain, weak but clear expression of hoxd13 was detected. C:Hoxd11 expression. The expression pattern was similar to the pattern in the forelimb (compare with 2C, 3C). D:Tbx3 expression. Expression of tbx3 was present in both the anterior and posterior margins.E:Sox9 expression. The first differentiating digit cartilage was observed (arrow). T; tibia (tibiale). F; fibula (fibulare). F-J: Gene expression in the forelimb bud at the digit stage (Nieuwkoop and Faber stage 54). F:Hoxa13 expression. Hoxa13 expression remained throughout the autopodium.G:hoxd13 expression. In the anterior domain, weak but clear expression of hoxd13 was still detectable (arrows in G,G'). H:Hoxd11 expression. Arrows in H and H' indicate the expression border. I:Tbx3 expression. Arrows in I indicate the expression border. J:Sox9 expression. The cartilage of putative digit I was just starting to express sox9 (arrow in J'). F'-J': Higher magnification of each figure. II-V are the digit numbers suggested by our results. Distal is to the top, and anterior is at the left.
Figure 3. Gene expression in Xenopus developing forelimb. A-E: Gene expression in the forelimb bud at the paddle stage. A:Hoxa13 expression. B: Hoxd13 expression. Hoxd13 expression in the posterior region was stronger than that in the anterior region. C:Hoxd11 expression. Hoxd11 expression was detected in the autopodium. The expression domain in the autopodium was restricted to the subectodermal region. Hoxd11 was detectable in the anteriormost region of the autopodium (arrow). D:Tbx3 expression. Expression of tbx3 was present in both the anterior and posterior margins. E:Sox9 expression. R, radius; U, ulna. The first differentiating digit cartilage was observed (arrowheads). F-J: Gene expression in the forelimb bud at the digit stage. F:Hoxa13 expression. The distal region of each digit still expressed hoxa13 at this stage. G:Hoxd13 expression. The hoxd13 expression pattern was similar to the hoxa13 expression pattern.H:Hoxd11 expression. Hoxd11 expression was detected in the anteriormost digit (arrows). I:Tbx3 expression. Arrowheads in I' indicate the expression border. J:Sox9 expression. Sox9 expression was detectable in digit cartilages. A'-J': Higher magnification of each figure. Arrowheads in I' indicate the tbx3 expression border. II-V are the digit numbers suggested by our results. Distal is to the top, and anterior is at the left. Roman numbers in F-J represent putative digit numbers.
Figure 4. Skeletal patterns of normal limb and spike. A: Normal forelimb of the froglet. B: Forelimb spike. C: Normal hindlimb of the froglet. D: Hindlimb spike. A comparison of forelimb and hindlimb spikes shows that they have similar structures. The numbers in parentheses are the numbers of phalanges. Broken lines in B and D indicate the amputation site.
Figure 5. Skeletal preparation of hindlimb regenerate and normal limb bud treated with cyclopamine. A: Lateral view of cyclopamine-treated limb bud and regenerate. Amputation was performed at the paddle stage (stage 52), and then samples were reared in 2 mu g/ml cyclopamine. B: Higher magnificent of A. C-F: Gene expression of cyclopamine-treated blastema. C:Hoxa13 expression was detected in the distalmost region (arrow). D:Hoxd13 transcription was also weakly detected (arrow). E:Hoxd11 expression was not observed in the cyclopamine-treated sample. F:Sox9 expression. The expression profile of these seems to be similar to that in a limb of the shh-deficient mouse.
Figure 6. Gene expression in regenerating froglet blastema. Section in situ hybridization was performed 2 weeks after amputation. A:Hoxa13 was expressed in the froglet blastema (arrowheads). B:Hoxd13 expression was also detected (arrowheads). C:Hoxd11 expression was detectable (arrowheads). D:Tbx3 expression. E:Sox9 expression. F: RT-PCR analysis for forelimb blastema and hindlimb blastema of the froglet. A-E: Distal is to the top, and anterior is at the right.
Figure 7. Digit amputation experiment. A: Dorsal view of regenerated digits. B: Higher magnification of A. C: Skeletal pattern of regenerated digit. D,E: Higher magnification of C. Regenerates were observed as spikes. No joint was observed in the digit spikes. Nails were observed in the anterior three spikes but not in the posterior two spikes. F: Spikes with a nail were formed in metacarpal-level amputation. White arrowheads indicate the nail. A,B: Asterisks mark joints, and white arrowheads indicate the nail. Lines in C-E indicate the amputation site.
Figure 8. Hoxa13 and hoxd11 expression in digit blastemas. A-D:hoxa13 expression. Hoxa13 expression was observed throughout the anteriormost blastema to the posteriormost blastema at 5 and 15 days after amputation. Hoxa13 expression was also detectable in the web region. E-G: Hoxd11 expression. E,F: At 5 days after amputation, hoxd11 expression was detectable throughout the anteriormost blastema to the posteriormost blastema. G: At 15 days, Hoxd11 was observed throughout the anteriormost blastema to the posteriormost blastema. However, weaker signals were detected in the anterior blastemas (arrowheads).