Decoding G0 somatic mutants through deep phenotyping and mosaic pattern analysis in the zebrafish skeleton

ABSTRACTGenetic mosaicism manifests as spatially variable phenotypes, whose detection and interpretation remains challenging. This study identifies biological factors influencing spatial phenotypic patterns in the skeletons of somatic mutant zebrafish, and tests methods for their analysis using deep phenotyping. We explore characteristics of loss-of-function clusters in the skeleton of CRISPR-edited G0 ("crispant") zebrafish, and identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. Using microCT-based phenomics, we describe diverse phenotypic manifestations in somatic mutants for genes implicated in monogenic (plod2 and bmp1a) and polygenic (wnt16) bone diseases, each showing convergence with germline mutant phenomes. Finally, we describe statistical frameworks for phenomic analysis which confers heightened sensitivity in discriminating somatic mutant populations, and quantifies spatial phenotypic variation. Our studies provide strategies for decoding spatially variable phenotypes which, paired with CRISPR-based screens, can identify genes contributing to skeletal disease.

Clonal expansion in the deciduous understory shrub, devil's club (Oplopanax horridus; Araliaceae)

To examine clonal growth and the ability of devil's club (Oplopanax horridus (Sm.) Torr. & A. Gray ex Miq.) to persist throughout stages of forest succession, we sampled populations in three classes of stand development: clearcuts (1–10 years old), young stands (11–50 years old), and maturing stands (51–200+ years old). We completely excavated and mapped all clonal fragments (systems of ramets connected by decumbent stems) in a plot at each site and determined ramet and clonal fragment age using annual growth rings on stem cross sections. Clonal fragment density and size, ramet density and age, decumbent stem length, annual stem elongation, annual ramet recruitment, lateral meristem formation, and the number of persistent dead ramets were significantly (P < 0.05) related to stand development class. Clonal fragments in clearcut stands were large, predating the year of stand establishment, with many dead, old ramets, but many young stems. Ramet recruitment and lateral meristem formation were highest in clearcut stands, which contributed to replacement of older ramets lost to the disturbance. Clonal fragments in young stands were few and small, consisting of a few ramets and short decumbent stems. In maturing stands, clonal fragments were numerous but consisted of few ramets with extensive decumbent stem connections. No devil's club seedlings were observed in any of the stands sampled. Devil's club populations are maintained by prolific basal stem sprouting following disturbance and continual layering and clonal fragmentation throughout stand development.Key words: age structure, clonal architecture, clonal shrubs, demography, layering, population dynamics.