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Science 10 October 1997:
Vol. 278. no. 5336, p. 305
DOI: 10.1126/science.278.5336.305
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Technical Comments

BMP Expression in Duck Interdigital Webbing: A Reanalysis

In 1996, two of us (H.Z. and L.N.) reported that expression of a dominant-negative form of BMP receptor (dnBMPR-IB) in the embryonic chick hindlimb inhibited interdigital apoptosis and led to webbing of the digits (1) (BMPs are signaling molecules of the transforming growth factor-betasuperfamily). The importance of BMP signaling in regulating interdigital cell death has recently been confirmed by the use of an activated BMPR-IB retrovirus (2) and by application of BMP protein (3, 4).

It was also stated in this report (1) that BMP2, 4, and 7 RNA expression was not detected in the duck interdigit. This result implied that the webbing in the hindlimb of ducks is a consequence of the absence of BMP expression in the duck embryo. After publication of the report (1), to explore this issue further, subsequent in situ hybridizations were carried out with the use of a modification (5) of an existing whole mount, in situ protocol. Results from our two different laboratories now indicate that BMP2, 4, and 7 are in fact expressed in the duck interdigit in a pattern similar to that of the chick interdigit (Fig. 1). The in situ protocol we used, in contrast with the protocol followed in the original study (1), included use of (i) a higher proteinase K concentration (30 to 70 µg/ml rather than 5 µg/ml), (ii) BCIP/NBT (6) as a color detection substrate rather than Boehringer-Mannheim purple AP substrate, and (iii) a TWEEN-20 concentration of 1% rather than 0.1% during the color substrate reaction. In combination, these modifications resulted in a greater sensitivity in detecting interdigital expression of BMP2, 4, and 7 in late-stage embryonic limbs. This result has been confirmed for BMP7 by nonradioactive in situ hybridization to frozen tissue sections (7). Therefore, we (H.Z. and L.N.) must withdraw the earlier finding that the duck interdigit lacks BMP expression and regret any inconvenience the earlier conclusions may have caused.

Fig. 1. Expression of BMP2, 4, and 7 in the interdigital regions of chick and duck limbs. Expression of each gene was detected by whole mount in situ hybridization to stage 28/29 and to stage 31 chick (8) and stage-equivalent duck embryos, as indicated. In each case, wing and leg buds were removed from the embryos after completion of the whole mount procedure and photographed from a dorsal view. [View Larger Version of this Image (38K GIF file)]

Ed Laufer
Department of Genetics,
Harvard Medical School,
Boston, MA 02115, USA
Sandrine Pizette
Hongyan Zou
Program in Molecular Biology,
Memorial Sloan-Kettering Cancer Center,
New York, NY 10021, USA
Olivia E. Orozco
Department of Genetics,
Harvard Medical School
Lee Niswander
Program in Molecular Biology,
Memorial Sloan Kettering Cancer Center

REFERENCES AND NOTES

  1. H. Zou and L. Niswander, Science 272, 738 (1996) [Abstract] .
  2. H. Zou, R. Wieser, J. Massagué, L. Niswander, Genes Devel. 11, 2191 (1997).
  3. Y. Yokouchi et al., Development 122, 3725 (1996) [Abstract] .
  4. D. Macias et al., ibid. 124, 1109 (1997) [Abstract].
  5. Whole mount in situ hybridizations were performed as described by R. D. Riddle et al. [Cell 75, 1401 (1993)], except that the temperature of all hybridizations and post-hybridization washes was 60°C and the proteinase K (PK) conditions were varied. Standard 1× PK is nominally 10µg/ml for 15 min at room temperature. However, we have noted batch variation in the specific activity of PK, and the conditions must therefore be titrated for each batch. Typically, 1× PK treatment results in the removal of most or all of the signal from s22 limb bud AERs, and strong mesenchymal signal for genes such as BMP2 or Sonic hedgehog. Maximal AER staining is usually seen around 1/4× PK. Older embryos often require substantially more PK treatment to reveal strong mesenchymal signals (up to 10× for 9-day-old embryonic limbs). In the experiments described here, PK conditions were independently optimized for visualization of interdigital BMP expression in both chick and duck limb buds. Following PK digestion, stage-matched chick and duck embryos were combined into a single vial and treated together for the remainder of the protocol. NBT/BCIP reactions were performed at room temperature for varying amounts of time (typically 1.5 to 3 hours), until a strong interdigital signal was observed. All of the probes were derived from the chick BMP genes, as described in Zou and Niswander (1).
  6. BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitro blue tetrazolium; both available from Sigma.
  7. E. Laufer et al., unpublished data.
  8. V. Hamburger and H. L. Hamilton, J. Exp. Morphol. 88, 49 (1951).
20 August 1997; accepted 9 September 1997


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