The limbs are highly variable structures between different mammalian and vertebrate species  enabling them to adapt and exploit new habitats. The vertebrate limb has served as a key model for understanding the signalling pathways controlling patterning and morphogenesis . Limb patterning genes and pathways have been well described in mice and chickens, but very few other model animals have been examined [1, 3, 4].
The tammar wallaby, Macropus eugenii, like all macropodid marsupials, has large hindlimbs specially adapted for hopping. The digits on the hindlimb are highly modified: digit 1 is never present, but digits 2 and 3 are fused and there is an elongated digit 4 . The tammar delivers an altricial young which climbs to the pouch using its relatively well developed forelimbs, but the hindlimbs are not yet functional and are essentially fetal. After birth, this situation changes and the hindlimb growth and development rapidly overtakes that of the forelimb during early pouch life. Despite this difference in timing, the tammar hindlimb autopod is specified before birth and the early formation of the syndactylous hindlimb digits is already initiated. However, the gene(s) regulating this process are as yet unknown.
Patterning of the vertebrate limb is coordinated by morphogens secreted across three different axes in the early limb bud; proximal distal (PD), dorsal ventral (DV) and anterior posterior (AP) [reviewed by ]. These morphogenic gradients dictate the formation of the stylopod (shoulder), zeugopodium (radius and ulna) and autopodium (hand and digits) [reviewed by ]. Fibroblast growth factor 8 (FGF8) expressed in the Apical ectodermal ridge (AER) controls a positive feedback signal that dictates PD outgrowth . Bone morphogenetic protein 4 (BMP4) is a key regulator of interdigital webbing and dorsal ventral polarity  while sonic hedgehog (SHH) secreted from zone of polarising activity (ZPA) (a signalling centre in the posterior region of the limb bud) acts via a negative feedback loop with BMP4 to determine anterior posterior patterning and digit identity ,
Interestingly, many of these genes and proteins also play conserved roles in the development of another appendage, the phallus [10, 11]. SHH is secreted from the urethral epithelium of the phallus and regulates patterning much like the ZPA in the limb bud [11, 12]. BMP4 is expressed in the phallus in the distal region near the urethral epithelium and interacts with SHH [reviewed by ]. Although these appendages are phenotypically different, the main signalling pathways remain the same  and Homeobox (HOX) genes underpin their regulation.
The HOX genes are crucial regulators of embryonic development and the phenotypic differences in the vertebrate body plan [reviewed by ] and are responsible for patterning the limb [reviewed by [12, 14, 15]]. Homeobox A13 (HOXA13) and Homeobox D13 (HOXD13) are essential for formation of the autopod and digit patterning in the mouse . HOXA13 mutants have fused digits and there is no digit 1 (the most anterior digit) . HOXD13 mutations result in fusion of digits 3 and 4 and a localised delay in autopod ossification . HOXA13 and HOXD13 double heterozygous mutants have more severe limb and genital phenotypes compared to either individual gene mutation .
Similarly in humans, mutations in HOXA13 and HOXD13 result in limb and genital malformations such as synpolydactyly, polydactyly and hypospadias [17–19]. Many of these mutations are due to the expansion or reduction of a polyalanine tract in these two genes [20, 21], changing the biochemical conformation . Interestingly, the chicken and zebrafish have shorter polyalanine tracts and both possess highly modified appendages . It is still not clear how the HOX genes are regulated  but human mutations such as Townes-Brocks  suggests that the transcription factors SALL1 and SALL3 influence the SHH and HOX pathways . Double null SALL1/SALL3 mutants have lost digit 1 and have fused digits 2 and 3, much like the HOXA13/HOXD13 double heterozygous mutants .
The bat Carollia perspicillata, like all chiropterans, has a highly specialised forelimb that evolved to enable flight . There is an expanded and posteriorly-shifted HOXD13 expression in the forelimbs compared to expression in the mouse [3, 26]. In addition, bats have developed a mechanism of interdigital retention involving the regulation of BMP4 and FGF8 through Gremlin (GREM1) . SHH has a second wave of expression in the bat forelimb  and may reinitiate the loop between FGF and SHH to retain the interdigital webbing of the bat and elongate the forelimb digits . In contrast, the macropodid marsupials have elongated and expanded their hindlimb digits, so a comparison of expression profiles for key limb patterning factors in the tammar may help to define the mechanisms underlying vertebrate limb diversity.
To date there is only limited information about the molecular control of marsupial limb development. The grey short-tailed opossum Monodelphis domestica has precocious forelimb development (as is also seen in the tammar) [1, 28] that facilitates the crawl from the birth canal to the mammary glands, but the hindlimb development lags slightly behind [29, 30]. This heterochronic development of the forelimbs is reflected by the timing of gene expression. Paired-like homeodomain transcription factor 1 (PITX1) expression an upstream inducer of T-box 4 (TBX4) is expressed late in opossum development relative to the mouse . TBX4 and T-box 5 (TBX5) are markers of hindlimb and forelimb position respectively and in the opossum are expressed relatively early in development, indicating that the opossum forelimb field arises relatively earlier than in the mouse and earlier than the opossum hindlimb [29, 31]. However, unlike the tammar, the opossum limbs are not especially modified and have all 5 digits .
We therefore focussed on the spatio-temporal changes of two genes, HOXA13 and HOXD13, known to be essential for digit development in the mouse and chicken, to investigate the possible role of HOX genes during digit and limb development in the tammar and to determine whether differential patterning accounts for their unique digit modifications.