Faculty Bibliography

Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation.

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

Stem cells maintain tissues and organs over the lifespan of individuals. How aging influences this process is unclear. Here we investigate the effects of aging on C. elegans germline stem/progenitor cells and show that the progenitor pool is depleted over time in a manner dependent on inhibition of DAF-16/FOXO by insulin/IGF-1 signalling (IIS). Our data indicate that DAF-16/FOXO activity in certain somatic gonad cells is required for germline progenitor maintenance, and that this role is separable from the effect of DAF-16/FOXO on organismal aging. In addition, blocking germ cell flux, similar to reducing IIS, maintains germline progenitors. This effect is partially dependent on gonadal DAF-16/FOXO activity. Our results imply that (1) longevity pathways can regulate aging stem cells through anatomically separable mechanisms, (2) stem cell maintenance is not necessarily prioritized and (3) stem cell regulation can occur at the level of an entire organ system such as the reproductive system.

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.