A dynamic and expandable digital 3D-atlas maker for monitoring the temporal changes in tissue growth during hindbrain morphogenesis.

Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a digital 3D-atlas maker (DAMAKER) and built a digital 3D-atlas to monitor the changes in the growth of the neuronal differentiation domain in the zebrafish hindbrain upon time. DAMAKER integrates spatial and temporal data of cell populations, neuronal differentiation and brain morphogenesis, through in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a 3D-reference from several imaged hindbrains and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation growth patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain by in vivo neuronal birthdating experiments. We generated digital neuronal birthdating 3D-maps and revealed that the temporal order of neuronal differentiation prefigured the spatial distribution of neurons in the tissue, with an inner-outer differentiation gradient. Last, we applied it to specific differentiated neuronal populations such as glutamatergic and GABAergic neurons, as proof-of-concept that the digital birthdating 3D-maps could be used as a proxy to infer neuronal birthdate. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction.

The brain, like most other organs, is formed by the coordinated growth of a few unspecialized cells in the embryo, which give rise to billions of neurons. For the brain to work properly, it is crucial that, during embryonic development, each neuron ends up in the correct location. This migration to the right spot has to happen while the brain grows and changes shape, which affects how and how far neurons and their precursor cells need to move to reach their final position. If these movements and changes in shape are not coordinated correctly, neurons can end up in the wrong place, form the wrong connections, and ultimately impact how the brain works. Previous work done in fruit flies and zebrafish resulted in three-dimensional maps of these animals' healthy brains, which allowed scientists to have a holistic view of how brains are organized. Although these maps are a valuable resource to study the structure of the brain, they do not provide information on how the brain transforms over time, especially during embryonic development. To get a clearer picture of how a few precursor cells give rise to the incredibly complex tissue that is the brain, a three-dimensional map spanning the entire developmental process is needed. To fill this gap in knowledge, Blanc et al. developed a digital atlas-maker pipeline (DAMAKER) that allows scientists to generate three-dimensional models of the embryonic brain from microscopy images of several individuals. They then used this pipeline to construct a three-dimensional digital atlas of how a part of the brain called the hindbrain develops in the zebrafish embryo. First, they collected images of the hindbrain showing neurons born at different times and matched these images to the existing static maps. Next, DAMAKER was used to follow neurons from the time of their birth to their final location, allowing Blanc et al. to create a map showing where neurons born at different stages during development end up. This type of map allows users to accurately determine when different populations of mature neurons are born, which allows scientists to estimate when different defects in brain development might originate. Based on these data, Blanc et al. concluded that in zebrafish most of the cells that will end up forming the hindbrain acquire their specialized neuronal identities very early in development, between 24 and 48 hours post fertilization. These temporal maps of healthy hindbrains were then compared to maps of brains in which the birth of neurons was disrupted, thus changing the final number of neurons in the brain. This experiment showed that changing the number of neurons that are born early in development alters the final positions of neurons and the overall shape of the brain. Therefore, for the brain to grow to its correct size, there must be a balance between the number of unspecialized cells in the developing brain, and the rate at which these cells become neurons. The DAMAKER pipeline not only provides scientists with a tool to study neurodevelopmental disorders, but also serves as a method that can be adjusted to map growth and shaping of other organs.

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-ß inhibitors. TGF-ß inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.