Phenotyping of Klf14 mouse white adipose tissue enabled by whole slide segmentation with deep neural networks

Abstract

AbstractWhite adipose tissue (WAT) plays a central role in metabolism, with multiple diseases and genetic mutations causing its remodeling. Quantitative analysis of white adipocyte size is of great interest to understand physiology and disease, but previous studies of H&E histology have been limited to a subsample of whole depot cross-sections. In this paper, we present the deep learning pipeline DeepCytometer, that can segment mouse and human whole slides (≃40,000 cells per mouse slide on average) using an adaptive tiling method, correct for cell overlap and reject non-white adipocytes from the segmentation. Using quantile colour maps we show intra- and inter-depot cell size heterogeneity with local correlation; quantile estimates also suggest significant differences in population estimates from 75 whole slides compared to smaller data sets. We propose three linked levels (body weight BW, depot weight DW and cell area quartiles) for exploratory analysis of mouse Klf14 phenotypes in gonadal and subcutaneous depots. We find a rich set of phenotypes when stratifying by sex, depot and three genotype strata: (1) WTs/Hets with a Het father (Controls), (2) WTs with a Het mother, and (3) Hets with a Het mother (functional KOs or FKOs). Namely, at BW level, mean difference testing suggests that female FKOs are similar to Controls, but WTs with a Het mother are significantly larger. At DW and cell levels, linear models with interaction terms and BW or DW covariates, respectively, reveal phenotypes not shown by difference of means tests. For example, at DW level, gonadal and subcutaneous models are similar, and female FKOs have lower fat percentage than Controls due to both an offset and the DW/BW slope in the linear model. Meanwhile, female WTs with a Het mother have on average similar fat percentage to Controls, but as their slopes are close to zero, their DWs are uncorrelated to BW, suggesting that larger female WTs with a Het mother have lower fat percentage than smaller ones. In contrast to depot level, at cell level female gonadal phenotypes diverge from subcutaneous ones. Furthermore, male Controls and FKOs have similar average area values in subcutaneous depots, but area~DW slope flattening in FKOs suggests that larger DWs could be caused by cell size increase in Controls and by cell count increase in FKOs. Thus, DeepCytometer and associated exploratory analysis reveal new insights into adipocyte heterogeneity and phenotyping.