Role of barhl1 in amphbian cerebellum

Cerebellar granule neuron progenitors (GNPs) originate from the upper rhombic lip (URL), a germinative niche in which developmental defects produce human diseases. T-cell factor (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity, together with transcripts encoding proneural gene repressors hairy and enhancer of split (Hes/Hey), are detected in the URL; however, their functions and regulatory modes are undeciphered. Here, we established amphibian as a pertinent model for studying vertebrate URL development. The amphibian long-lived URL is TCF active, whereas the external granular layer (EGL) is non-proliferative and expresses hes4 and hes5 genes. Using functional and transcriptomic approaches, we show that TCF activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL, acting partly through direct TCF inhibition. Identification of Barhl1 target genes suggests that, besides TCF, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role for Barhl in maintenance of the URL and/or EGL via co-regulation of TCF, Hes and Hey genes. RNA- sequencing analysis was performed on dissected Rhombomere 1 from Xenopus Laevis tadpoles depleted or not for BarH-like 1 (Barhl1), a direct target gene of Atonal 1 (Atoh1), a master regulator of cerebellar glutamatergic neurons development, and an inhibitor of T-Cell factor activity. X. laevis embryos were injected with three different conditions: MObarhl1-1; MObarhl1-2 and MOct in the two dorsal blastomeres at four cells stage. At stage 42, neural tubes were extracted in RNAse-free conditions, and the rhombomere 1 which includes the URL was carefully dissected. For each condition, three biological replicates were collected. Each replicate contains three rhombomeres. Poly A RNA was purified. Sequencing was performed using Illumina NovaSeq (paired-end sequencing) by Next Generation Sequencing Platform (NGS) (Institut Curie, Paris FR). This transcriptomic analysis identifies Barhl1 cerebellar target genes, and confirms that Barhl1 acts as a gate keeper for progenitor exit from the URL, through silencing of T-Cell Factor transcriptional activity.