Supporting Online Material for

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1 Supporting Online Material for Embryological Evidence Identifies Wing Digits in Birds as Digits 1, 2, and 3 Koji Tamura,* Naoki Nomura, Ryohei Seki, Sayuri Yonei-Tamura, Hitoshi Yokoyama *To whom correspondence should be addressed. tam@m.tohoku.ac.jp This PDF file includes: Materials and Methods Figs. S1 to S7 References Published 11 February 2011, Science 331, 753 (2011) DOI: /science

2 Materials and Methods Animal Procedures. Eggs of white Leghorn chicken (G. gallus) and quail (C. japonica) were purchased from several local farms, incubated at 38 C, and staged according to Hamburger and Hamilton (S1) and Ainsworth et al. (S2). Gecko embryos were obtained from individuals of P. pictus, which were hatched and kept in our laboratory, and the embryos were staged according to Noro et al. (S3). Mouse embryos were collected at various gestation times from pregnant mice (M. musculus) that were also obtained from local suppliers and staged following Theiler (S4). Transplantation experiments. A small piece of tissue was excised from the center ZPA (see Fig. S1 and the legend) and implanted into a slit under the AER made at the anterior margin of a host stage 20/21 limb bud using sharpened tungsten needles. In some cases, the ectoderm of graft tissue was removed by trypsin before implantation; the results obtained were the same as from the grafts with ectoderm. For the analysis of skeletal pattern, embryos seven days after the above manipulations were fixed in Tyrode s solution containing 10% formalin, stained in 0.1% alcian blue, dehydrated in graded alcohols, and cleared in methyl salicylate. Quail-chick chimera. Chick (host) and quail (donor) embryos were used for the analysis of chimeras to determine the distribution of graft tissue in host limb buds. Tissue fragments from the quail limb buds were implanted into chick limb buds, as described above. After 5-6 days, excised limbs were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) at 4 C for 12 hours, washed in PBS at 4 C for 3 hours, and immersed in 10% and 20% sucrose PBS for 6 hours and overnight, respectively. Samples were embedded in O.C.T. compound (Miles), frozen in liquid nitrogen, and sectioned at 10μm using a cryostat (Leica). The QCPN monoclonal antibody (the Developmental Studies Hybridoma Bank), a quail-specific antibody, was used for examining the distribution of grafted quail tissues. Dye injection. Administration of DiI (1,1-dioctadecyl-3,3,3,3 -tetramethyl indocarbocyanine perchlorate; Molecular Probes) and DiO (3,3 -dioctadecyloxacarbocyanine perchlorate; Molecular Probes) was performed as described previously (S5). Dye administration by pressure of expiration was performed using a pulled micropipette with an open tip. In order to determine the position and area injected with dye, dye-labeling was performed under a fluorescence microscope, and the labeled limb buds were photographed immediately after dye administration. The anterior-posterior position of the dye spot were calculated with the aid of a micrometer. At 5 6 days after the above process, operated limb buds were re-photographed. Whole-mount in situ hybridization. Mouse, chick, and gecko embryos were processed for whole mount in situ hybridization, as described respectively (S3, S6, S7). Riboprobes for chicken shh, fgf8, aggrecan1 (proteoglycan-h), gecko shh and fgf8, mouse shh and fgf8 were generated as described previously (S3, S5, S6, S7).

3 Supplementary figures Figure S1. Scheme of experimental procedures. (A-C) Swapping transplantation of the ZPA at stages 21/22 and 25. A single graft tissue from the center of the ZPA (the middle region of shh expression, region outlined in D F outside the posterior edge (yellow arrowheads) of the AER) was excised from a donor limb bud at each stage. The same region as that used for transplantation was also used for homotopic chimera analysis in Figure S2 (orange and blue-outlined cube). The two pink dots in D and E are the points for fate mapping of the region inside and outside the ZPA at stage 22 in Figure 2.

4 Figure S2. Chimera analysis for fate-mapping of stage 22 ZPA. (A-D, I, J) Chick forelimb buds 6 days after the central region of the ZPA homotopically replaced with quail tissue. (C, D) show details from (A, B) at higher magnification. Few cells can be seen in the cartilage of the posteriomost digit (asterisks), and the quail ZPA are seen scattered in connective tissue surrounding the cartilage (n=7). Another example of forelimb ZPA

5 chimera shows the contribution of ZPA cells in a small cartilage in the wrist (asterisks in I, J). (E-H) Hindlimb buds six days after the central region of the ZPA had been replaced with quail tissue. (G, H) show details from (E, F) at higher magnification. Metatarsi and phalanges (asterisks) of the posteriormost digit are composed of ZPA-derived cells (n=5). In summary, forelimb ZPA transplanted at stage 22 did not contribute to digit cartilage; the descendants of the ZPA cells were instead scattered in connective and dermal tissues in the posterior peripheral region of the forelimb autopod and zeugopod. Descendants of ZPA cells did however contribute to the formation of a small, rounded cartilage in the base of the forelimb autopod, which may represent a rudimentary P5-derived cartilage (fifth condensation of digit primordium shown at bottom panel in Fig. 4). In the hindlimb, the homotopic ZPA descendants generated the digit D4.

6 Figure S3. Fate maps based on labeling experiments at stage 20. (A, B, D, E) Independent specimens of DiI/DiO labeling of stage 20 forelimb (A, B) and hindlimb (D, E) buds. Relative position of the injected point is indicated to the left in (A, D). (A) Two points at 15.7% and 4.7% were labeled in this sample. The labeled cells at 15.7% contributed to digit D3, and cells at 4.7% were involved not in a digit but in the extreme periphery in (B). (C) Diagram of the fate of the posterior forelimb bud at stage 20. Points that contributed to digits D2 and D3 and posterior periphery (PP) are indicated by pink, orange, and blue circles, respectively. Black circles indicate the average position of each color (32.6% for pink (n=11), 17.7% for orange (n=15), and 6.6% for blue (n=26)). Yellow circles indicate the anterior margin of the shh-expression domain, and the average position is at 21.4% (n=12). (D) Two points at 12.3% and 3.4% are labeled in this sample. The labeled cells at 12.3% were involved in digit D4, and the labeled cells at 3.4% were distributed to the posterior periphery in (E). (F) shows a diagram of the fate of the posterior hindlimb bud at stage 20. Black circles indicate the average position of each color (36.2% for pink (n=12), 18.9% for orange (n=22), and 6.6% for blue (n=16)). Yellow circles indicate the anterior margin of the shh-expression domain, and its average position at 23.1% (n=12). Figure S4. Separation of digit 3 progenitor from the ZPA in the forelimb bud. Labeling of anterior and middle ZPA. (A, B) Forelimb bud. In this sample, a point at 17.5% (inside shh-expressing domain, see Fig. 3 and Fig. S3) was labeled at stage 20. After 18 hours, the labeled cells were seen outside the shh-expressing domain. (C, D) Hindlimb bud. In this sample, an anterior point inside the ZPA (at 21.6%) was labeled at stage 20. After 18 hours, the labeled cells were seen inside the shh-expressing domain. (E, F) Labeled cells at 15.5% of stage 20 forelimb bud (E) were located outside the shh-expressing domain after 18 hours (F). Labeling of posterior ZPA is shown in Fig. 3, C and F.

7 Figure S5. Overlapping zone of the ZPA and the AER. All images are magnified, and focused on the posterior distal region of developing limb buds. (A-C) Chick forelimb buds. (D F) Chick hindlimb bud. Stage 22 (A, D). Stage 24 (B, E). Stage 26 (C, F). (G I) Mouse forelimb buds. E10.5 (G). E11 (H). E11.5 (I). (J-L) Gecko forelimb buds. 6 days postoviposition (dpo) (J). 9 dpo (K). 12 dpo (L). These mouse and gecko limb buds morphologically correspond to chick stage 22, 24, and 26 limb buds. Note that the domain of overlap between the AER and the ZPA (distance between arrowhead and red dot) is correlated with digit numbers of chick forelimb (three digits), chick hindlimb (four digits), and mouse/gecko forelimbs (five digits). The early reduction of the overlap

8 in the chick forelimb might be involved in heterochronic shift of digit anlagen. Red dots show the anterior margin of the shh-expression domain. Arrowheads show the posterior edge of the fgf8-expression domain. Scale bar, 100 μm. Figure S6. Artificial posteriormost digit reallocated on the primary axis. (A) Image representation of posterior removal of a stage 22/23 hindlimb bud. Red broken arrows indicate approximate position of tissue removal (ZPA and the AER being visualized by shh and fgf8expression). (B) A sample resulting from the removal in (A). Only the anterior two digits (D1 and D2) form (n=8/10). (C) Mesenchymal condensation after posterior removal, visualized by expression of aggrecan 1. The second digit (digit D2) is located on the primary axis (yellow arrow). (D) Hindlimb of contralateral side of (C) as a control, showing the fourth digit (digit D4) located on the primary axis (yellow arrow). Ti, Tibia. Fi, Fibula. Similar results have also been obtained with Cyclopamine inhibition of Shh signaling (S8)

9 Figure S7. Contributions of fore- and hindlimb ZPAs to duplicated digit cartilage. Some typical examples are shown in Fig. 1. (A) Table showing donor-type digit in forelimb-hindlimb transplantation of the ZPA. (B) A specimen of forelimb ZPA to

10 hindlimb, which has a small forelimb digit-like digit (arrowheads). This sample is categorized in with a forelimb digit. (C) A specimen of forelimb ZPA to hindlimb, which has a small unidentified digit (arrowheads). This sample is categorized in with an unidentified digit. (D) A specimen of hindlimb ZPA to forelimb, which has an extra-segment at the distal tip of the anteriormost digit (arrowheads). This digit may represent the hindlimb digit, but it is vague. Therefore, this sample is categorized in only forelimb digits. Supplemental References S1. V. Hamburger, H. L. Hamilton, J. Morphol. 88, 49 (1951). S2. S. J. Ainsworth, R. L. Stanley, D. J. Evans, J. Anat. 216, 3 (2010). S3. M. Noro et al., Dev. Dyn. 238, 100 (2009). S4. K. Theiler, The house mouse. Atlas of embryonic development (Springer-Verlag, New York, 1989). S5. K. Sato et al., Development 134, 1397 (2007). S6. S. Yonei-Tamura et al., Dev. Biol. 211, 133 (1999). S7. S. Yonei-Tamura et al., Evol. & Dev. 10, 737 (2008). S8. A. O. Vargas, G. P.Wagner, Evol. & Dev. 11, 163 (2009).