A handful of neural progenitors give rise to thousands of diverse neuronal cell types that perform complex functions. These ?blank slate? progenitors? transition through a complex molecular network to become functionally and spatially distinct mature neurons. However, the molecular networks that drive neuronal fate decisions are poorly understood. One important group of progenitors express the proneural transcription factor, Atonal homolog 1 (Atoh1), and originate at the rhombic lip (RL) region of the hindbrain. Atoh1 progenitors are defined by spatial location at early stages of development and migrate away from the RL in distinct migration streams to ultimately give rise to all cerebellar excitatory neurons and dozens of brainstem nuclei responsible for critical functions (e.g. balance, hearing, and breathing). Failure of Atoh1 progenitors to properly form these distinct nuclei can be detrimental to life. Despite how important the Atoh1 lineage is to health and disease, the molecular network that directs Atoh1 progenitors through stages of differentiation remains unknown. If the transcriptional trajectories of Atoh1 progenitors were elucidated, regenerative therapies could be developed to repair genetic aberrations that lead to improper development, which would reduce the burden of neurological disease. The long-term objective of this proposal is to elucidate the molecular networks that drive the neuronal diversity of Atoh1 progenitor development to improve therapeutics to restore function following trauma to the central nervous system. The hypothesis of this proposal is that Atoh1 progenitors undergo temporal and spatial fate decisions driven by a defined molecular network. The objectives of this proposal are to identify the gene or sets of genes that drive neuronal fate decisions in the Atoh1 lineage and to determine if transcriptional trajectories are conserved in in vitro models of hindbrain development.
Specific Aim 1 will test the hypothesis that a molecular cascade drives Atoh1 lineage diversity by instructing progenitors when, where, and how to differentiate. The molecular cascade will be determined by isolating the Atoh1 lineage from embryonic stage 9.5 to 18.5 and performing single cell RNA sequencing (scRNAseq). In situ hybridization will be used to identify spatial distribution of transcripts and confirm scRNAseq results.
Specific Aim 2 will test the hypothesis that in vitro-derived mouse hindbrain organoids undergo similar transcriptional trajectories to in vivo development. A stem cell-derived mouse hindbrain model will be developed by exogenously adding agonists of endogenous signaling morphogens. Organoid composition and comparison to in vivo mouse hindbrain development will be elucidated though scRNAseq. The collective results will add to the fundamental knowledge of nervous system development by elucidating the molecular network that drives development of Atoh1 progenitors in a critical brain region. The strategies used in this proposal would be broadly applicable to studying progenitor development in other brain regions.
One common neuronal progenitor in the hindbrain gives rise to over 40 different neuronal nuclei responsible for breathing, hearing, arousal, and balance. Disruption of these nuclei due to developmental disorders, injury, or cancer is detrimental to life. To work towards solutions to restore or prevent neurological dysfunction in the hindbrain, the proposed project will uncover the molecular network that drives fate commitment from one common progenitor to diverse mature neurons using a combination of mouse genetics and stem cell-derived hindbrain models.
