Douglas B. Menke - US grants

DESCRIPTION (provided by applicant): Tbx4 and Tbx5 are critical for hindlimb and forelimb development, respectively. The developmental regulation of these transcription factors in forelimbs and hindlimbs of mice will be elucidated, and downstream targets of these genes will be identified. Using comparative genomics and mouse transgenics, a hindlimb enhancer has recently been localized to a 5.5kb region of Tbx4. The minimal portion of this sequence that can direct hindlimb expression in transgenic mice will be identified. Experiments will also be performed to isolate cis-regulatory elements that direct the forelimb expression of Tbx5. Candidate trans-acting factors that may act upon these cis-regulatory elements will be identified using comparative genomics and yeast-one hybrid screens, and it will be established whether these candidate factors are directly involved in the regulation of Tbx4 and Tbx5 limb expression. Finally, genes that are differentially expressed between embryonic forelimbs and hindlimbs will be isolated, and it will be determined whether these genes are direct downstream targets of Tbx4 or Tbx5. Collectively, the identification of both upstream regulators and downstream targets of Tbx4 and Tbx5 will help reveal the precise roles that these genes play in forelimb and hindlimb development.

Animal species display tremendous diversity in body size and shape, yet relatively little is known regarding the genetic mechanisms responsible for these differences. The objective of this project is to examine this question by determining whether species that exhibit similar limb lengths acquire these comparable limb morphologies through related developmental mechanisms. Anolis lizards are ideal for these studies since they display remarkable differences in limb length between species. During the course of this project the pattern of limb growth and gene expression will be compared between multiple different long-limbed and short-limbed Anolis species. In addition, Anolis species with naturally occurring differences in DNA sequences that control hindlimb formation will be investigated to determine whether these sequence differences can directly alter the function of limb genes. These developmental and molecular analyses will reveal whether different species with similar body forms achieve a similar final appearance through the same or different means. The proposed studies will also generate broader impacts through the creation of new teaching and training opportunities for undergraduate, graduate, and postdoctoral scholars, as well by providing resources that will advance Anolis lizards as a new model system for developmental studies. Finally, the project will integrate research and education through a research module that has been developed for an advanced undergraduate molecular genetics laboratory course.

DESCRIPTION (provided by applicant): Development of the limbs and external genitalia require the coordination of many different regulatory pathways and signaling events. While it is now clear that important components of the limb and genital appendage gene networks are shared, remarkably little is known about the cis- regulatory elements that contribute the transcriptional control of these networks. The Tbx4, Pitx1, and Isl1 transcription factors are among a very small number of hind limb-specific genes that function in the hind limb but not the forelimb. Though work in animal models and humans has shown a central role for these factors in leg formation, we have almost no knowledge of the functional interactions these transcription factors make with the network of cis-regulatory elements that promote limb development. In addition, our preliminary data indicates that at least of one these factors, Tbx4, is also an important regulator of genital growth, suggesting that these genes may exhibit overlapping functional interactions with appendage cis-regulatory elements active in the hind limbs and genitals. The objective of this proposal is to determine how Pitx1, Tbx4, and Isl1 influence the development of appendages, by investigating their interactions with the suite of cis- regulatory elements active in the developing hind limbs and genitalia. This objective will be accomplished by 1) identifying the set of enhancer elements that are bound and directly regulated by Pitx1 in the hind limbs and genitals, 2) determining whether Tbx4 directs the outgrowth of the legs and external genitalia via similar mechanisms, and 3) establishing whether the initiation of hind limb bud and genital bud formation is regulated by Isl1 through a common set of regulatory interactions. The knowledge gained from these studies will help to achieve our long-term goal of understanding how the disruption of specific regulatory interactions can lead to malformations of the limbs and genitals in humans.

Natural selection, the differential survival and reproduction of individuals possessing different traits, is a major process driving evolutionary change. One sign of natural selection is convergent evolution: when traits in different species that occupy the same environment evolve in a similar matter. Although convergence was once assumed to be rare, recent research demonstrates that convergence occurs quite commonly. An outstanding question, however, is whether closely related species and populations are more likely to converge, both in their physical characteristics and in their genes, than more distant relatives. Similarities in natural history and genetic makeup might predispose close relatives to adapt in similar ways to similar circumstances. Moreover, contemporary adaptation to the changed environmental circumstances resulting from human actions is increasingly common. It is not known whether species are adapting to anthropogenically induced selective pressures in the same way as they have adapted to natural changes in the environment. This is an important question because species' survival in the face of environmental change may depend on evolving adaptations to new conditions. Conservationist scientists are gaining interest in how to make evolutionary adaptation more likely. The predictions about adaptation that will result from this research may be useful in guiding actions to maximize that evolutionary potential of populations. Students will be trained in international scientific research, and several outreach activities will engage the general public in better understanding of scientific issues.

This study will address these questions by focusing on two groups of lizard, Caribbean Anolis lizards and South African chameleons, and examining the degree to which convergence occurs at multiple levels: between lizard groups separated by millions of years of evolutionary divergence, within each of these clades, and among modern-day populations adapting to human-altered environments. These questions will be investigated both by examining anatomical characteristics, for example leg length and body size, and by studying genetic differences among diverse groups of lizards. In addition, comparisons across populations and species will help identify candidate genes that may be responsible for the observed anatomical differences. New gene editing methods will be used to directly test whether these genetic changes actually produce the predicted anatomical changes. These studies will play an important role in understanding the limits of natural selection in shaping adaptation to future environmental change.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.