Primers designed for this study, which were used in sequencing and in QPCR for the three limb development genes and ribosomal 28S in Pogona vitticeps from Expression of a hindlimb-determining factor <i>Pitx1</i> in the forelimb of the lizard <i>Pogona vitticeps</i> during morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the Central Bearded Dragon (<i>Pogona vitticeps</i>) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (<i>GHR, Pitx1</i> and <i>Shh</i>) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimb and hindlimbs of <i>P. vitticeps.</i> While the highest level of <i>GHR</i> expression occurred at the hatchling stage, <i>Pitx1</i> and <i>Shh</i> expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of <i>Pitx1</i> expression—a hindlimb-determining gene—in the forelimbs of <i>P. vitticeps</i> to that in a closely related Australian agamid lizard, <i>Ctenophorus pictus</i>, where we found <i>Pitx1</i> expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent to that found across other tetrapods. Expression of <i>Pitx1</i> in forelimbs has only rarely been documented, including via <i>in situ</i> hybridization in a chicken and a frog. Our findings, from both RT-qPCR and IHC, indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology.