Reza Kalhor

The Johns Hopkins University School of Medicine, USA

Organ  development  is  guided  by  a  space-time  landscape  that constrains cell fate. This landscape is challenging to characterize for the hair follicle – the most abundant mini organ – due to its complex microscopic structure and asynchronous development. We developed 3DEEP,  a  tissue  clearing  and  spatial  transcriptomic  strategy  for characterizing tissue blocks up to 400 µm in thickness. We captured 371 hair follicles at different stages of organogenesis in 1 mm3 of skin of a 12-hour-old mouse with 6 million transcripts from 81 genes. From

this  single  time  point,  we  deconvoluted  follicles  by  age  based  on whole-organ  molecular pseudotimes to animate a stop-motion 3D atlas of follicle development along its trajectory. We defined  molecular  stages  for  hair  follicle  organogenesis  and  characterized  the   order  of emergence for its structures, differential signaling dynamics at its top and bottom, morphogen shifts preceding and accompanying structural changes, and series of structural changes leading to the formation of its canal and opening. We further found that hair follicle stem cells and their niche are established and stratified early in organogenesis, before the formation of the hair bulb. Overall, this work demonstrates the  power  of increased  depth  of spatial transcriptomics to provide a four-dimensional analysis of organogenesis.