Achaete-Scute-Like 1a Expression Is Essential for the Generation of Correct Proportions of Neuron Subtypes in the Developing Enteric Nervous System of Zebrafish (Danio rerio).

BACKGROUND AND AIMS: The enteric nervous system (ENS) mediates many gastrointestinal functions and develops through processes involving transcription factors, including achaete-scute-like 1a (Ascl1a). In zebrafish, the contribution of Ascl1a to enteric neuronal specification remains unclear, which we aim to unravel.

METHODS: We examined spatiotemporal Ascl1a expression in the ENS of wild-type (WT) zebrafish larvae using quantitative polymerase chain reaction (qPCR) and immunofluorescence staining. Ascl1a function was investigated using pia t25215 zebrafish (Ascl1a -/- ). Immunofluorescence staining was performed to identify and quantify (immature) enteric neurons and neuronal subtypes.

RESULTS: A peak in Ascl1a mRNA expression was detected at 3.0 days postfertilization (dpf) in the gastrointestinal tract of WT larvae. In their ENS, the proportion of Ascl1-expressing neurons declined between 2.5 and 4.0 dpf. In contrast, ratios of Ascl1+ neural progenitors and immature neurons remained stable between 3.0 and 4.0 dpf. Ascl1a deletion resulted in delayed and suppressed onset of ENS development, with neurons appearing only at 4.0 dpf in significantly lower numbers than in WT larvae. Additionally, neuronal subtype composition was altered with a significant increase in serotonergic and a significant decline in calbindin-expressing neurons, the latter showing delayed emergence at 5.0 instead of 3.0 dpf. Nitrergic neuron ratios were unaffected.

CONCLUSION: Ascl1a deletion resulted in delayed and partially suppressed ENS development and altered neuronal subtype populations; the calbindin-expressing neuron ratio was significantly reduced, while that of serotonergic neurons was significantly increased. This indicates Ascl1a involvement in both early and late enteric neuronal differentiation and specification.