The lateral line system in fish is crucial for detecting water flow, which facilitates various behaviors such as prey detection, predator avoidance, and rheotaxis. The cupula, a gelatinous structure overlaying the hair cells in neuromasts, plays a key role in transmitting mechanical stimuli to hair cells. However, the molecular composition of the cupula matrix remains poorly understood. In this study, we found that Mucin-5AC, a novel family of mucin proteins, composed of 2–27 cysteine-rich domains, presents in cartilaginous and bony fishes. Using in situ hybridization and transgenic reporter assays, we demonstrated that zebrafish muc5AC is specifically expressed in the support cells of neuromasts. Knockdown of muc5AC via antisense morpholino resulted in shorter cupulae in zebrafish lateral line. Additionally, we generated zebrafish muc5AC mutants using CRISPR/Cas9 and found that cupulae in muc5AC mutants were significantly shorter than that in wild-types, but the hair cell number in neuromasts was not changed obviously. Furthermore, muc5AC mutant zebrafish larvae displayed compromised sensitivity to vibration stimuli compared to wild-type larvae. This study provides the first evidence linking the muc5AC gene to cupula development and vibration detection in zebrafish. Our findings suggest that Mucin-5AC is likely a critical component of the cupula matrix, offering an important clue to the molecular composition of the lateral line cupula in fish.

Signal transducer and activator of transcription 3 (STAT3) is essential for neural development and regeneration as a key transcription factor and mitochondrial activator. In this study, using zebrafish posterior lateral line (PLL) axons, we demonstrate that Stat3 plays distinct roles in PLL axon embryonic growth and regeneration. In zebrafish stat3 mutants the PLL axons were truncated during embryonic growth. Expressing stat3 in neurons of the PLL ganglion rescued their axon growth suggesting stat3 promoted PLL axon growth in a cell-autonomous manner. Using Jak/Stat signaling inhibitors or modified Stat3, we found Jak/Stat signaling was dispensable for PLL axon growth. We showed that Stat3 was co-localized with mitochondria in PLL axon and lower ATPase activity in stat3 mutant embryos than wild-type embryos. The PLL axon growth was affected by mitochondrial modulators indicating that Stat3 regulated PLL axon growth through mitochondrial Stat3 function. Mutation of stat3 or treating with Jak/Stat signaling inhibitors retarded PLL axon regeneration and Schwann cell migration. Taken together, Stat3 is required for embryonic PLL axon growth by regulating the ATP synthesis efficiency of mitochondria, whereas Stat3 stimulates PLL axon regeneration through regulating Schwann cell migration via Jak/Stat signaling. Our findings show a new mechanism of Stat3 in axon growth and regeneration. Author Summary: Neurons are one of the most specialized cell type in our body. A slender axon used for transmitting information between neuron and target cell extends from the cell body. Axon extension during both development and regeneration is regulated by intrinsic and extrinsic factors. In this study, we used the zebrafish posterior lateral line (PLL) axons which extends a long distance to study the regulation of axon extension. Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor in Jak/Stat signaling and an activator for mitochondria functions. Our results demonstrate that Stat3 plays distinct roles in PLL axons development and regeneration. During embryonic development, Jak/Stat signaling is dispensable for PLL axons extension, whereas the mitochondrial Stat3 is required for embryonic axon growth through promoting ATP synthesis. During PLL axons regeneration, Jak/Stat is essential for Schwann cells migration which mediates axon regeneration.