Weatherbee, S. D. | bioRxiv Science

bioRxiv2018

Presence of midline cilia supersedes the expression of Lefty1 in forming the midline barrier during the establishment of left-right asymmetry

Cilia in the vertebrate left-right organizer are required for the original break in left-right (L-R) symmetry. Subsequently, proper L-R patterning relies on asymmetric expression of Nodal in the lateral plate mesoderm (LPM). Lefty1, expressed in the embryonic midline, has been defined as the midline barrier, restricting the expression of Nodal to the left LPM. Here we use the mouse ciliary transition zone mutant Mks1krc that has left isomerism and bilateral expression of the NODAL target Pitx2, to reveal that the expression of Lefty1 in the midline is insufficient for the establishment of the midline barrier. We further show through a comparison of two Tmem107 mutants that cilia in the midline are required to supplement Lefty1 expression and establish the functional midline barrier. Tmem107null mutants have no cilia in the midline and display left isomerism due to the loss of the midline barrier, whereas Tmem107schlei hypomorphic mutants have numerous cilia in the node and the midline, leading to normal Lefty1 expression and L-R patterning. This study reveals a novel role for cilia in the maintenance of L-R asymmetry.

developmental biology↗

bioRxiv2017

Disrupting Pitx1 Regulatory Topology Results In Overtly Normal Limb Development

Gene expression patterns during development are orchestrated in part by thousands of distant-acting transcriptional enhancers. However, identifying enhancers that are essential for expression of their target genes has proven challenging. Genetic perturbation of individual enhancers in some cases results in profound molecular and developmental phenotypes, but in mild or no phenotypes in others. Topological maps of long-range regulatory interactions may provide the means to identify enhancers critical for developmental gene expression. Here, we leveraged chromatin topology to characterize and disrupt the major promoter-enhancer interaction for Pitx1, which is essential for hindlimb development. We found that Pitx1 primarily interacts with a single distal enhancer in the hindlimb. Using genome editing, we deleted this enhancer in the mouse. Although loss of the enhancer completely disrupts the predominant topological interaction in the Pitx1 locus, Pitx1 expression in the hindlimb is only reduced by ~14%, with no apparent changes in spatial distribution or evidence of regulatory compensation. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1-/- mutant. Our results indicate that Pitx1 expression is robust to the loss of its primary enhancer interaction, suggesting disruptions of regulatory topology at essential developmental genes may have mild phenotypic effects.

genetics↗