Wu, J. P. H. | bioRxiv Science

bioRxiv2021

Temporal analysis of enhancers during mouse brain development reveals dynamic regulatory function and identifies novel regulators of cerebellar development.

In this study, we identified active enhancers in the mouse cerebellum at embryonic and postnatal stages establishing the first catalog of enhancers active during embryonic cerebellum development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Our study provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.

neuroscience↗

bioRxiv2020

The transcription factor Pou3f1 provides a new map to the glutamatergic neurons of the cerebellar nuclei

Pou3f1 is a transcription factor involved in early neural differentiation. Cap Analysis Gene Expression (5-CAGE) analysis reveals that Pou3f1 transcript is highly enriched in the developing cerebellum. Between embryonic (E) days E10.5 and E12.5, Pou3f1 expression is present prominently along the subpial stream (SS), suggesting that Pou3f1+ cells are glutamatergic cerebellar nuclear (CN) neurons. This finding was confirmed by immunofluorescent (IF) co-labeling of Pou3f1 and Atoh1, the master regulator of cells from the rhombic lip (RL) that are destined for neurons of the glutamatergic lineage, as well as in Atoh1-null tissues, in which Pou3f1 expression is absent. Interestingly, the expression of Pax6, another key molecule for CN neuron survival, does not co-localize with that of Pou3f1. In the Pax6-null Small Eye (Sey) mutant, which is characterized by a loss of many glutamatergic CN neurons, Pou3f1+ CN neurons are still present. Furthermore, Pou3f1-labeled cells do not co-express Tbr1, a well-established marker of glutamatergic CN neurons. These results highlight that Pou3f1+ cells are a distinct and previously unrecognized subtype of glutamatergic CN neurons that do not have the "canonical" sequence of Atoh1[->]Pax6[->]Tbr1 expressions. Instead, they express Atoh1, Pou3f1, and other markers of CN neurons, Brn2 and Irx3. These findings illustrate that glutamatergic CN neurons that arise from the RL are composed of molecularly heterogeneous subpopulations that are determined by at least two distinct transcriptional programs. Significance StatementThe present work has identified Pou3f1 as a marker for a previously unidentified subtype of glutamatergic cerebellar nuclear neurons, the principal output neurons of the cerebellum. The classical model of glutamatergic CN neurons development follows the sequential expression of transcription factors Atoh1[->]Pax6[->]Tbr1. However, we found that the development of Pou3f1+ neurons requires Atoh1 but not Pax6. Moreover, Pou3f1+ neurons do not express Tbr1, but instead express two other transcription factors, Brn2 and Irx3. Anatomically, Pou3f1+ CN neurons populate the interposed and dentate nuclei, while the Tbr1+ CN neurons populate the fastigial nucleus. These findings reveal the heterogeneity of CN neuron populations and the diversity of molecular programming that lead to different CN neuron subtypes.

neuroscience↗