Forward Genetics, Cilia, and Limb Development

Abstract

Our research is focused on identifying genes and characterizing biological mechanisms that regulate organ formation and patterning during embryonic development. To identify key developmental and disease genes, we use powerful and unbiased forward genetics screens, which have become feasible due to recent advances in high-throughput sequencing. In the course of our studies, we have identified three mouse lines with mutations in cilia genes (Tmem107, Ttc26, Mks1). Cilia are microtubule-based, membrane-enclosed organelles found on the surface of almost all vertebrate cells and have been identified as key regulators of animal development and disease. The establishment of distinct digit identities is essential for the proper functioning of the limb and Sonic Hedgehog (Shh) signaling plays the major role in specifying the number and identity of digits. Recently, Shh signaling in vertebrates has been shown to be dependent upon the cilium. First, Shh pathway components including the receptor Ptch1, the positive pathway mediator Smo, and even the transcriptional mediators of the pathway, the Gli proteins, localize to the cilium. Second, mutations in genes that are required for cilia formation or maintenance cause Shh signaling defects in the embryo. Within the limb, cells with defective cilia generally do not perceive the Shh signal and lose the expression of genes downstream of Shh signaling. Surprisingly, the limbs of mouse cilia mutants do not resemble a Shh loss of function phenotype, but instead develop extra digits, i.e. polydactyly. Work from several labs has shown that cilia are required to form functional Gli-Activator (GliA). Cilia are also required to generate the processed repressor (GliR) forms, indicating that cilia are required for both the activator and repressor activities of Gli proteins. Loss of cilia or abnormal cilia function often results in polydactyly in mouse models and polydactyly is a defining feature of many cilia-related diseases (ciliopathies). We found that our cilia mutants (Tmem107, Ttc26, Mks1) appear to specifically affect the function of GliR in the limb. In addition, we have uncovered a late role for cilia in the regulation of programmed cell death in the limb. We are working to further define the genetic network that regulated cilia formation and function during limb development. Given the breadth and severity of cilia-associated defects, elucidating their underlying genetics is critical for diagnostics and defining the molecular function of cilia genes is essential to understanding how this small organelle impacts animal development and human health. Support or Funding Information R01AR059687 from NIAMS/NIH, P30DK090744 from NIDDK/NIH, training grant T32GM007499 from NIGMS/NIH, and training grant T32HD007149 from NICHD/NIH, and National Science Foundation Graduate Research Fellowships under DGE-0644492 and DGE-1122492. Disrupting cilia gene function causes polydactyly (asterisks). (A) Control, (B) Mks1 mutant, and (C) Tmem107 mutant E18.5 hindlimbs. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.