Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum

Abstract

A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip.

The generation of granule cells during the development and evolution of the cerebellum

“…They discovered that the early atoh1c ‐expressing isthmic progenitors gave rise to anterior hindbrain neurons outside the cerebellum, likely analogous to the neurons derived from an early isthmic domain of atoh1 expression described in the chick . Atoh1c RL progenitors give rise to granule neurons as expected . Interestingly the deletion of atoh1c eliminated most but not all granule neurons in the cerebellar corpus, homologous to the mammalian cerebellar vermis, however atoh1a ; atoh1c double mutants lacked all GC neurons in the corpus.…”

“…Zebrafish possess three Atoh1 genes: atoh1a , atoh1b , and atoh1c . They are all expressed in the anterior hindbrain during the formation of the cerebellar primordium, with important differences in nonoverlapping domains . Atoh1a and atoh1b are broadly expressed, encompassing the entire rhombic lip and hindbrain, with the former being more strongly expressed.…”

The interplay of atoh1 genes in the lower rhombic lip during hindbrain morphogenesis

“…atoh1a fh282 mutant line in the Tg[atoh1a:GFP] background, which carried a missense mutation within the DNA-binding domain, was previously described in [18]. Embryos were phenotyped blind and later genotyped by PCR using the following primers: Fw primer 5 0 -ATGGA TGGAATGAGCACGGA-3' and Rv primer 5 0 -GTCGTTGTCAAAGGCTGGGA-3'.…”

“…The three atoh1 paralogs -atoh1a, atoh1b and atoh1c-were shown to be expressed within the hindbrain and to contribute to the development of the cerebellum, with the expression of atoh1c restricted to the upper rhombic lip [17,18]. Since our main interest was understanding the development of the lower rhombic lip (LRL), we focused on the study of atoh1a and atoh1b and compared their onset of expression.…”

“…The rest of the RL, which expands from r2 to r7, is known as Lower Rhombic Lip (LRL) and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei [16,17]. The genetic program for cerebellum development is largely conserved among vertebrates [16]; as an example, zebrafish and mouse use similar mechanisms to control cerebellar neurogenesis with a crucial role of atoh1 and ptf1 genes [17,18]. For the LRL, we know both the contribution of ptf1a/atoh1a proneural progenitor populations to specific deep nuclei [19], and the distinct rhombomeric identity [20].…”