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Figure 1: Xenopus laevis limb bud location and orientation, and Tschumi’s fate maps of the hind limb bud
mesenchyme. A) A stage 52 albino Xenopus laevis tadpole, viewed from left side, with anterior (head) to
the left and dorsal (back) uppermost. The positions of the limb buds are marked. Fl: forelimb bud, hl: hind
limb bud. B) Schematic of limb bud orientation. Viewed “naturallyâ€, in the same orientation as in (A), limb
buds are initially oriented with proximal to the left, posterior uppermost, and are seen from the dorsal
surface, with the ventral surface obscured. C) Re-drawings of Tschumi’s 1957 fate maps of the
mesenchyme of the Xenopus laevis hind limb (Tschumi, 1957), with a hind limb skeleton for comparison .
Stages (numbers 48 to 54) have been retrospectively assigned according to the commonly morphological
staging criteria for this species (Nieuwkoop and Faber, 1967). Note that, as the hind limbs develop, cells
fated to become progressively more distal structures of the limb were able be labelled. Roman numerals
relate to the forming digits, with digit I the most anterior. s= stylopod (upper limb long bones),
z= zeugopod (lower limb long bones) and a= autopod (hand/foot). Fate maps in C are re-drawn from
(Tschumi, 1957) with permission from Wiley publications.
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Figure 2: Evidence of a transient morphological AER in Xenopus laevis tadpole limbs. Plates from Figures 5
and 7 in Tarin and Sturdee 1971, reproduced with permission from Journal of Embryological Experimental
Morphology. Full article can be accessed here. A) Ventral longitudinal section through a stage 51 tadpole
showing the AER. Note that the epidermis has three layers at this point and the basal cells are clearly
columnar. The marginal sinus (V) is visible in the subajacent mesenchyme. Bar is 10μM. B) Scanning
electron micrograph of stage 51 hind limb viewed from the distal tip with dorsal suface to left. The AER runs
diagonally from left to right and extends for a short distance proximally on the dorsal and ventral surfaces
which slope away steeply in this picture. Contaminating particles marked with asterisks should be
disregarded. Bar is 20μM.
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Figure 3: Molecular marker data summary. Representative sketches showing markers commonly used in
limb bud morphogenesis studies, or of interest, in stages where the genes have been shown to be expressed
by whole mount in situ hybridisation or in sections. Orientation of the hind limb is ânaturalâ, as in Fig.1b,
proximal to left, posterior top and viewed from the dorsal surface. Note that, due to the dorsal
mesenchymal location of lmx1b, it is also shown viewed from the anterior. For lbx1, the presence of the
proximal posterior expression is assumed to be present but could not be determined from published data
due to the presence of obscuring melanophores. Abbreviations: RA: retinoic acid, AER; apical ectodermal
ridge, ZPA: zone of polarizing activity. Digit IV is marked for orientation in stage 54. Numbers at top
correspond to stages (Nieuwkoop and Faber, 1967). Data based on images in (Christen and Slack, 1997;
Endo et al., 1997; Endo et al., 2000; Christen and Slack, 1998; Matsuda et al., 2001; Satoh et al., 2005;
Martin and Harland, 2006; Satoh et al., 2006; Miura et al., 2008; Christen et al., 2012; Jones et al., 2013;
Wang and Beck, 2014)
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Figure 4: Schematic depicting a possible origin of the mesenchyme cells of Xenopus hind limb buds based on
A. Cecil Taylor’s 1943 observations in Rana pipiens. A partial frontal section, with the anterior of the tadpole
upwards, is represented at the level of the hind limb buds at E25 (approx stage 44 Xenopus). Taylor
observed the presence of specialised somatopleure cells (ss, black circles) in the region where the hind limbs
(hl) form. In this region the cells were atypical for somatopleure (s) cells, being rounded rather than
squalmous epithelial cells, and suggesting epithelial to mesenchymal transition (EMT). Between these cells
and the hind limb mesenchyme cells Taylor observed a region, which he called the “bridge†(b) of elongated
cells, aligned as if migrating from the ss to hl region and forming a connection between the two regions
which persisted at least until the limbs became capable of autonomous development (L1-L2 in Rana, 48 in
Xenopus). Other abbreviations: E; epidermis, f; fin, m, subdermal mesenchyme (somatic mesoderm of
lateral plate), c, coelom (body cavity); p, proctodeum (anal cavity). Adapted from (Taylor, 1943).
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Figure 5: The main blood vessels in the developing hindlimb buds of Xenopus laevis as shown by Tschumi,
1957. Note the large marginal vessel (mv) that has developed from the marginal sinus (Fig. 4a) and system
of radial capillaries in the autopod. Most capillaries in the proximal region have been omitted for clarity. A)
stage 52 B) stage 54, positions of digits are marked in roman numerals Figure from (Tschumi, 1957) is
reproduced with permission from Wiley publications.
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Figure 6: Components of the Xenopus laevis limb skeleton A) ventral view of stage 60 Xenopus laevis
metamorph stained to show ossified bone (red, alizarin red) and cartilage (blue, alcian blue), to show the
bones of the limb girdles, stylopod (upper limb), zeugopod (lower limb) and some parts of the autopod
(hand/foot). B) Schematic showing the arrangement of elements in the forelimb autopod and C) the hind
limb autopod. Note the elongated tarsus (tibiale and fibulare), lack of interclavicles, and that the ilium is not
fixed to the sacrum, all of which are typical of extant frogs. Also note the conserved phalangeal count of
forelimb (3,3,2,2) and hind limb (2,2,3,4,3). The designation of the forelimb digits as II-V, rather than I to
IV seen in older texts, follows the convincing molecular experimental and ontological evidence of (Satoh et
al., 2006). For clarity, not all tarsals and carpals are depicted.
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