## Why use the chick limb bud as a model system?

Humans have five distinct digits along the anterior-posterior (thumb to little finger) limb axis (Fig. 1). When we look at our hands and feet, we can see that the thumb/big toe is the widest and shortest among the five digits, while the little finger is the narrowest and smaller than the middle three digits, and the middle three digits – digits 2, 3, and 4 – appear similar to each other, although their morphology and length are slightly different. These morphological and anatomical differences between digits are called “digit identity”. Hands and feet develop from a small population of somatic lateral plate mesoderm cells, called the limb bud, which grows out from the body wall. The limb is characterized by three axes: the anterior-posterior (AP) axis described above, the proximal-distal (PD) axis (shoulder/thigh to fingers), and the dorsal-ventral (DV) axis (knuckles to palm). The study of digit identity is designed to uncover the mechanisms of AP axis determination in the limb bud. The limb skeleton is organized into three anatomical regions: (i) the stylopod (humerus/femur), (ii) the zeugopod (ulna and radius/tibia and fibula), and (iii) the autopod, which consists of carpal/tarsal bones, metacarpal/metatarsal bones, and phalanges (Fig. 1). Phalanges are classified into three types according to their position along the PD axis: proximal phalanx, middle phalanx, and terminal phalanx, which has nails or claws/hoofs. Humans have two phalanges in the thumb/big toe, a characteristic morphological criterion of digit 1 in all amniotes (Vargas & Fallon 2005a,b; Wagner & Vargas 2008; Woltering & Duboule 2010). The posterior four digits have three phalanges, so other criteria are necessary to distinguish each digit. The mouse has five digits, as well, and the morphological differences in each are similar to those in human digits, with the middle three digits (digits 2, 3, and 4) appearing similar (Fig. 2). To distinguish among digits 2, 3, and 4 in the mouse limb morphologically, it is necessary to assess metacarpal/metatarsal articulation in the carpals/tarsals at least after E16.5, when the ossified tip of the terminal phalanx begins to be visible (Verheyden *et al*. 2005). In the forelimb, the digit 2 metacarpal articulates with the trapezoid and central carpal bones. Similarly, the digit 3 metacarpal articulates with the capitate bone, and the digits 4 and 5 metacarpals with the hamate bone. The digit 1 metacarpal articulates with trapezium bone. In contrast, in the hindlimb, the digit 2 metatarsal articulates with the intermediate and navicular bones and is located proximal to the intermediate bone, while the digit 3 metatarsal articulates with the lateral cuneiform bone and the digit 4 metatarsal with the cuboid bone.

The avian forelimb is a highly derived structure that is modified for flight and is not representative of amniote limb development in general (Fig. 2). The avian wing has three digits that were thought to be digits 2, 3, and 4 for most of the last century. The reasons for this have been reviewed extensively recently (see for example, Young & Wagner 2011; Young *et al*. 2011 and will not be discussed here). However, Tamura *et al*. reported, using cell lineage analysis, that the chick wing has digits 1, 2, and 3; this finding fits nicely with paleontological findings (Tamura *et al*. 2011; see also Towers *et al*. 2011). Transcriptome data for each digit in the chick forelimb and hindlimb also suggest that gene expressions in digit 1 in the wing are similar to those of digit 1 in the leg (Wang *et al*. 2011). In contrast, the chick foot has four digits, which are easily distinguishable as digits 1, 2, 3, and 4 (Fig. 2). Compared with the chick wing, the chick leg is more closely representative of amniote limb structures and is a highly useful model for studying digit identity determination because of its anatomy.

Importantly, it is easier to identify each digit morphologically in the chick embryo than in the human or mouse. Digit identity is characterized by three major morphological criteria: number, size, and shape of the phalanges (Suzuki *et al*. 2008). All three criteria are necessary to judge each digit identity. To identify each digit by these criteria, first, it is easy to count phalanx numbers in the hindlimb. To establish this, we have to examine terminal phalanx formation, which occurs after St. 36 (Hamburger & Hamilton 1951). Similar morphological criteria should also be applied to the mouse digits. *Gli3*^{−/−} mice have unidentifiable digits because of their abnormal phalanx morphology (Litingtung *et al*. 2002). Therefore, the identities of digits in *Gli3*^{−/−} mice cannot be determined by morphological criteria, although attempts to determine digit identity in these mice have been reported (e.g., Lopez-Rios *et al*. 2012). With regard to caution about counting phalanx numbers, the chick hindlimb is an exceptionally useful model system for studying digit identity determination. In the hindlimb, digits 1, 2, 3, and 4 have two, three, four, and five phalanges, respectively, so this criterion is unambiguous and therefore easy to use as a first step (Fig. 2).