Bambi and Sp8 expression mark digit tips and their absence shows that chick wing digits 2 and 3 are truncated

Abstract

An often overlooked aspect of digit development is the special nature of the terminal phalanx, a specialized structure with characteristics distinct from other phalanges, for example the presence of ectodermal derivatives such as nails and claws. Here, we describe the unique ossification pattern of distal phalanges and characteristic gene expression in the digit tips of chick and duck embryos. Our results show that the distal phalanx of chick wing digit 1 is a genuine tip with a characteristic ossification pattern and expression of Bambi and Sp8; however, the terminal phalanx of digits 2* and 3 is not a genuine tip, and these are therefore truncated digits. Bambi and Sp8 expression in the chick wing provides a direct molecular assessment of digit identity changes after experimental manipulations of digit primordia. In contrast, digits 1 and 2 of the duck wing both possess true tips. Although chick wing-tip development was not rescued by application of Fgf8, this treatment induced the development of extra phalanges. Grafting experiments show that competence for tip formation, including nails, is latent in the interdigital tissue. Our results deepen understanding of the mechanisms of digit tip formation, highlighting its developmental autonomy and modular nature, with implications for digit reduction or loss during evolution. * Numbering of wing digits is 1, 2, 3 from anterior to posterior.

Figure 1. Ossification pattern of the terminal phalanx in chick digits.

A: Ossification in the tip of leg digit 3 on the indicated days of development, as detected by alizarin red staining. Note the ventral initiation of ossification (arrowhead) and the progression towards the distal end. A proximal phalanx (phalanx 1 of digit 3, P1D3, at E10), with an evident central ossification ring, is shown for comparison (arrows). The bottom right panel shows a stained longitudinal section in which alizarin red is evident at the distal extreme of the tip phalanx; other panels show whole-mount specimens. B: Ossification of the terminal phalanges of chick wing digits. Ossification in digit 1 is similar to that in leg tips, starting ventrally and progressing distally (arrowheads). In contrast, the ossification of digit 2 resembles a proximal/intermediate phalange, with the presence of a central ossification ring (arrows).

Figure 2. Expression of Sp8 and Bambi in digit tips.

A: Sp8 in situ hybridization in mouse digits at the indicated developmental stages. (a-g) Whole-mount hind limbs. Note that Sp8 is expressed in the AER up to E13 (a,b, arrows) and later reappears in the tip ectoderm (d-f). Expression is stronger dorsally (g) and examination of stained paraffin sections shows that it is restricted to the ectoderm (h). B: In situ hybridization for Sp8 in chick digits at the indicated Hamburger and Hamilton stages. Sp8 is expressed in the AER of all leg digits up to HH32 (a,b, arrows) and later reappears in the tips of digits (d-f). Expression is limited to the tip, showing a sharp boundary (g), and is restricted to the ectoderm (h, paraffin section). (i) In the wing, Sp8 is expressed at the tip of digit 1 (arrow) but is absent from the ends of digits 2 and 3. C: Bambi in situ hybridisation in chick digits at the indicated stages (a-e, legs; f-j, wings). Note the strong initial expression in the interdigital space mesenchyme (a, f, arrows) that subsequently fades away, and the expression in the digit tips as the last phalanx forms. This terminal expression is observed in all toes (e) but is only present in the tip of digit 1 in the wing (j). Within digits, Bambi is sharply restricted to the tip (k) and is expressed only in the ectoderm (l, paraffin section). Bambi is also expressed (arrows) in leg scales (m) and feather buds (n,o).

Figure 3. Digit 2 of the duck wing has a genuine tip.

A: The pattern of ossification (alizarin red staining) of the last phalanx of digits 1 and 2 is distal (arrows). B: (a) Fresh wing specimen, showing the evident claw at the end of digit 2 (arrow). (b,c) In situ hybridisation showing Bambi expression at the tips of duck digits at E13. (b) Expression is detected in wing digits 1 and 2 (arrowheads). (c) In the leg, Bambi is expressed in all toe tips.

Figure 4. Bambi tip expression marks changes of digit identity triggered by surgical manipulations.

Two surgical manipulations were performed in HH27 wings to transform the identity of digit 2 towards digit 1. Seven days after the operation, Bambi expression was detected by in situ hybridisation. A: In type I experiments, the posterior part of the digit 2 primordium and the posterior interdigital space 2 were removed (see scheme in the inset in a). (a) The remnant digit 2 primordium has developed into a shorter digit (transformed digit 2*) with a tip positive for Bambi expression (arrow). (b) Magnified view of Bambi expression in the tip of digit 1 in the operated limb. (c) Bambi-negative tip of digit 2 in the control (non-operated) wing. (d) Bambi expression in the tip of the operated and transformed digit 2*. B: In type II experiments the digit 2 primordium was bisected (see scheme in the inset in a). (a) The extra digit formed from the anterior half of the digit 2 primordium (transformed digit 2*) shows expression of Bambi in the tip (arrow). (b) Magnified view of Bambi expression in the tip of digit 1 in the operated limb. (c) Bambi-negative tip of digit 2 in the control wing. (d) Bambi expression in the tip of the operated and transformed digit 2*. C: Digits from the specimen in A (and additionally the digit 1 from the control wing) were sectioned after the Bambi in situ hybridisation and stained with haematoxylin & eosin to detect transformation of cartilage elements. The length of phalanx 2 (P2) relative to phalanx 1 (P1) in transformed digit 2* is reduced, resembling the proportions in digit 1. (e) Magnified view of the boxed area in d (rotated 90°). Note the expression of Bambi in the tip ectoderm of the transformed digit 2* (arrows).

Figure 5. Fgf8 induces elongation and extra phalanges in digits 1 and 3, but not digit 2, of the chick wing.

Beads soaked in Fgf8 (1 mg/ml) were applied to the first (B,C) or second (D,E) interdigital spaces of HH27 wings. Five days after the operation embryos were collected and stained with alcian green to reveal skeletal elements. A: Scheme showing the position of the beads at the time of operation. Text refers to the panels showing each experiment. B, C: Application to the first interdigital space induced elongation of digit 1 (B, arrow), sometimes with the production of a complete extra phalange (C, arrow) with a new joint (asterisk). In both cases, the tip is normal (arrowheads). D: Application to the second interdigital space did not induce elongation of digit 2, but caused widening (D, arrow). E: In contrast, this treatment induced elongation of digit 3 with the formation of an extra phalanx (arrow) with a new joint (asterisk). Operated wings are shown on the right in panels B,C and on the left in panels D,E (all dorsal views).

Figure 6. Interdigital tissue can form ectopic digits with tips and claws.

Explants of distal interdigital space 2 (2ID: A,B) or distal digital ray 3 (D3: C,D) were taken from HH27 legs (distal 150 µm plus AER) and grafted to the somites of HH20 hosts. 2ID and D3 explants both formed ectopic digits with phalanges (alcian blue staining, A,C) and claws (white keratinized area in fresh specimens, B,D), detected 8–10 days after transplantation.