Keenan SR and Beck CW (2016), Xenopus Limb bud morphogenesis.

XB-ART-51289

Dev Dyn 2016 Mar 01;2453:233-43. doi: 10.1002/dvdy.24351.

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Xenopus Limb bud morphogenesis.

Keenan SR , Beck CW .

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Xenopus laevis, the South African clawed frog, is a well-established model organism for the study of developmental biology and regeneration due to its many advantages for both classical and molecular studies of patterning and morphogenesis. While contemporary studies of limb development tend to focus on models developed from the study of chicken and mouse embryos, there are also many classical studies of limb development in frogs. These include both fate and specification maps, that, due to their age, are perhaps not as widely known or cited as they should be. This has led to some inevitable misinterpretations- for example, it is often said that Xenopus limb buds have no apical ectodermal ridge, a morphological signalling centre located at the distal dorsal/ventral epithelial boundary and known to regulate limb bud outgrowth. These studies are valuable both from an evolutionary perspective, because amphibians diverged early from the amniote lineage, and from a developmental perspective, as amphibian limbs are capable of regeneration. Here, we describe Xenopus limb morphogenesis with reference to both classical and molecular studies, to create a clearer picture of what we know, and what is still mysterious, about this process.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 cyp26a1 cyp26b1 fgf8 gdf5 itk kcnt1 lbx1 lmx1b.1 lmx1b.2 shh sox9 tbx2 zp2

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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 “naturally”, 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. 99x57mm (300 x 300 DPI)
	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. 175x85mm (300 x 300 DPI)
	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 “natural”, 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) 145x121mm (300 x 300 DPI)
	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). 78x73mm (300 x 300 DPI)
	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. 86x88mm (300 x 300 DPI)
	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. 175x139mm (300 x 300 DPI)