Evolution of the ZRS and the Regulation of SHH Expression in the Forelimbs of Bats.

Brito, Denise Ribeiro Arthur

Evolution of the ZRS and the Regulation of SHH Expression in the Forelimbs of Bats.

Thesis / Dissertation

2011

Abstract

The characterisation of sonic hedgehog (Shh) in the developing limbs of the two bat species Miniopterus natalensis and Carollia perspicillata showed that Shh expression pattern was different from that observed in mouse limbs. An interesting discovery was that bat embryos display a second round of expression at stage CS16/E12.5. The aim of this thesis is to investigate the cause of the Shh expression pattern in developing limbs of the bat. Sonic hedgehog expression in the limb is regulated by a long-distance cis-regulatory sequence called the ZPA-regulatory sequence (ZRS). Mutations in the ZRS are known to result in limb deformities in humans, mice and cats. I investigated whether conserved mutations occurring in the bat ZRS are the cause of the change in Shh expression in bat limbs during embryonic development. Comparison of the ZRS of eighteen bat species to the ZRS of twenty other vertebrates revealed five conserved mutations that are unique to the bat ZRS. To test if the bat ZRS has the ability to change the expression pattern of Shh, the ZRS from M. natalensis and Rhinolophus clivosus (which have different wing shapes) was linked to a β-globin-LacZ reporter construct and used in transgenic mice experiments. A mouse ZRS with bat mutations as well as a wild type mouse ZRS (the control) were also tested. Transgenic results suggest that the bat ZRS has the elements necessary to alter the spatial expression pattern of Shh, but that may not be able to induce a second round of expression. The two bat ZRS reporter construct species produce different β-galactosidase expression patterns - a result of having very different ZRS sequences - thus implying that there is interspecies variation of the Shh expression pattern. Phylogenetic analysis of bat ZRS grouped the bat species in three well supported clades, which may not only reflect differences in Shh expression but also differences in wing shape. To test this hypothesis, it was first necessary to identify which skeletal elements within the handwing vary the most across the bat species, and consequently account for the variation in wing shape. Analysis of wing morphology revealed that phalange I of digits 3 and 4, and phalange II of digits 3, 4 and 5 showed significant variation across 53 bat species, along with Aspect ratio, which is an indicator of wing shape. Comparisons of Aspect ratio between bat species in the ZRS clades imply a relationship between differences in the ZRS sequences of bats and wings. Analysis of the variation of lengths of five skeletal elements mentioned above between the ZRS clades suggest that the wing shape is determined by different combinations of the length of 3PII, 3PI, 4PI, 4PII, and 5PII. This suggests that the variation of the skeletal elements may be a result of post-natal development as juvenile bats learn to fly, or that there is another mechanism determining wing shape.

Keywords

Molecular and Cell Biology

Reference:

Collections

Masters

Full item page